Methods and compositions that affect melanogenesis

ABSTRACT

The invention provides methods of screening for compounds that affect melanogenesis and the function of P protein in organisms, cells, or cell-free systems. The invention further relates to pharmacologic and cosmetic uses of methods of inhibiting melanogenesis, methods of activating melanogenesis, and compounds and pharmacologic compositions useful for the inhibition or activation of melanogenesis and, therefore, for lightening or darkening the pigmentation of cells and tissue, i.e., skin.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/599.487, filed Jun. 23, 2000, which claims priority fromU.S. Provisional Application Ser. No. 60/141,563, filed Jun. 29, 1999,both of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grantnumber EY10223 awarded by the U.S. Public Health Service. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1 Field of the Invention

[0004] The invention relates to the fields of medicine and cell biology.More specifically, the invention relates to the fields of drug discoveryand dermatology, particularly the biology of skin pigementation.

[0005] 2 Description of Related Art

[0006] Melanin is a dark pigment found in plants and animals thatprotects against ultraviolet radiation and provides decoration in theskin, eyes, hair, and fur of animals (reviewed in Riley, P. A., 1997,Int. J. Biochem. Cell Biol. 11:1235-39). There are two different typesof melanin: brown/black eumelanin and yellow/red pheomelanin.Melanocytes are cells of the epidermis specialized to produce melanin. Asophisticated intercellular signaling system determines whether anindividual melanocyte will produce eumelanin or pheomelanin (reviewed inBrilliant, M. H. and Barsh, G. S., 1998, in The Pigmentary System:Physiology and Pathophysiology, 217-29, Oxford University, New York(Nordlund, J. J. et al, eds)).

[0007] Melanocytes synthesize melanin inside of specialized organellescalled melanosomes (reviewed in Orlow, S. J., 1998, in The PigmentarySystem: Physiology and Pathophysiology, 97-106, Oxford University, NewYork (Nordlund, J. J. et al, eds)). Melanosomes are formed by the fusionof two types of vesicles. One type of vesicle, called a premelanosome,apparently derives directly from either the smooth endoplasmic reticulumor the trans-Golgi network. The other type of vesicle derives from thetrans-Golgi network. Each of these types of vesicles contributesproteins to the melanosome necessary for its function.

[0008] Defects in the production of melanin result in pigmentationdeficiencies such as albinism. Genetic analysis of abnormally pigmentedstrains of mice has identified more than 60 genes necessary for thenormal production of melanin (reviewed in Silvers, W. K., 1979, The CoatColors of Mice: A Model for Mammalian Gene Action and Interaction,Springer-Verlag, Basel). One of these genes encodes the enzymetyrosinase. Tyrosinase protein is a multi-functional enzyme thatcatalyzes several steps in the production of melanin; tyrosinaseactivities include the rate-limiting steps of converting tyrosine todihydroxyphenylalanine (DOPA), and DOPA to dopaquinone (reviewed inLerner, A. B., and Fitzpatrick, T. B., 1950, Physiol. Rev. 30:91-126),as well as the oxidation of 5,6-dihydroxyindole to 5,6-indolequinone(Korner and Pawelek, 1982, Science 217:1163-1165). Both humans and micelacking tyrosinase activity suffer a severe form of albinism.

[0009] Two tyrosinase-related proteins (TRP-1, encoded by the mousebrown gene, and TRP-2, encoded by the mouse slaty gene) also areimportant for melanogenesis (reviewed in Hearing, V. J., 1993, Am. J.Hum. Genet. 52:1-7). Each of the TRP proteins shares about 40% sequenceidentity with tyrosinase and with each other. Each of these threeenzymes (tyrosinase, TRP-1 and TRP-2) is predicted to contain onetransmembrane domain. Together, they form a high molecular weightcomplex associated with the melanosomal membrane (Orlow, S. J., et al.,1994, J. Invest. Dermatol. 103:196-201).

[0010] Another protein that is important for the production of melaninis the P protein. In mice, it is the product of the pink-eye dilution(p) gene. In humans, it is the product of the P gene. p-null miceproduce significantly less melanin than wild-type mice (Silvers, supra).A wild-type human P gene, but not a mutant human P gene, can complementthe hypopigmented phenotype of p-null mouse melanocytes (Sviderskaya, E.V., et al, 1997, J. Invest. Dermatol 108:30-34). P protein is apparentlyneeded for the production of eumelanin, but not of pheomelanin(Lamoreux, M. L., et al., 1995, Pigment. Cell. Res. 8:263-70).

[0011] Tyrosinase positive oculocutaneous albinism (Ty-pos OCA) or type2 oculocutaneous albinism (OCA2) is the most common form of albinismworldwide. It results from mutations at the pink-eyed dilution gene (P)(King, R. A. et al (1995) Scriver, C. R. et al. (eds) The MetabolicBasis of Inherited Disease, McGraw-Hill, New York. pp 4353-4392; Ramsay,M. et al (1992) Am. J. Hum. Genet. 51:879-84; Rinchik, E. M. et al.(1993) Nature 361: 72-76). Affected individuals have hypopigmented skin,hair and eyes (Manga P. et al (1999) J. Dermatol. 26:738-47), and arethus at increased risk of developing UV-induced carcinomas (Kromberg, J.G. et al. (1989) Clin. Genet. 36:43-52).

[0012] The p gene product (p), is predicted to have 12 transmembranedomains (Gardner, J. M. et al. (1992) Science 257:1121-1124; Rinchik, E.M. et al. (1993) Nature 361:72-76), and is present in melanocytes, thesite of melanin synthesis (Rosemblat, S. et al. (1994) Proc. Natl. Acad.Sci. (USA) 91:12071-12075). While p appears to be involved exclusivelyin eumelanin synthesis (Russell, E. S. (1949) Genetics 34:146-166), aprecise function is yet to be assigned to the gene.

[0013] Several authors have suggested that P protein acts as a tyrosinetransporter by pumping tyrosine into the melanosome where it isconverted into melanin by tyrosinase activity (see, e.g., Rinchik, E.M., et al., 1993, Nature 361:72-76). First, the P protein bears someresemblance to transport proteins found in prokaryotes. Second, culturedp-null mutant mouse melanocytes, which produce much less melanin thancultured wild-type mouse melanocytes, make increased levels of melaninwhen high concentrations of tyrosine are added to the cells growthmedium (Sviderskaya, E. V., et al., supra; Rosemblat, S. et al., 1998,Exp. Cell Res. 239:344-52). However, contradicting this suggestion, ithas been found that tyrosine uptake by melanosomes is virtually the samein p-null and wild-type melanocytes (Gahl, W. A. et al., 1995, Pigment.Cell. Res. 8:229-233). This observation has led other authors tohypothesize that P protein is necessary for the transport intomelanosomes of some other small molecule necessary for melanogenesis(summarized in Brilliant, M. H. and Barsh, G. S., 1998, supra).

[0014] Other authors have speculated that P protein plays a structuralrole in melanosomes (Lamoreux, M. L., et al., supra). The integrity ofmelanosomes is compromised in cells lacking P protein. Tyrosinaseactivity, and therefore melanin production, is greatly decreased inthese defective melanosomes. Specifically, tyrosinase activity levels inmelanocyte extracts of skin and eyes from p-null mice are lower thansuch extracts from wild-type mice (Lamoreux, M. L., et al., supra Chiu,E., et al, 1993, Exp. Eye Res. 57:301-05). Moreover, levels oftyrosinase, TRP-1 and TRP-2 proteins are lower in p-null tissue extractsthan in wild-type extracts (Rosemblat, S. et al., 1998, supra).Additionally, a much greater percentage of tyrosinase, TRP-1, and TRP-2proteins are found in their monomeric forms, rather than as part of ahigh molecular weight complex, in p-null tissue extracts than inwild-type extracts (Lamoreux, M. L., et al., supra; Chiu, E., et al.,supra), and tyrosinase, TRP-1, and TRP-2 are all rapidly degraded in theocular tissue of p-null mice (Chiu, E., et al., supra). Finally, severalauthors have observed that melanosomes in p-null tissues and culturedmelanocytes are abnormal (Russell, E. S., 1949, Genetics 34:146-66;Rosemblat, S. et al., 1998, supra). In p-mutant melanocytes from mouseeye, very few melanosomes are observed (Orlow, S. J. and Brilliant, M.H., 1999, Exp. Eye Res. 68:147-54). In cultured mutant melanocytes, agreater than normal number of melanosomes is present, but they aresmaller than those seen in wild-type melanocytes (Rosemblat, S. et al.,1998, supra).

[0015] Thus, although P protein is known to be critical for theproduction of normal amounts of melanin in the skin, hair and eyes, thefunction of the P protein in this process has remained elusive. Instead,researchers have looked to other molecular targets for inhibitionstudies. For example, tyrosinase's well-characterized enzymaticactivity, amenability to biochemical analysis, and pivotal role inmelanogenesis have made it an inviting target for inhibition studies(see, e.g., Tasaka, K., et al., 1998, Meth. Find. Exp. Clin. Pharmacol.20:99-109; lida, K., et al, 1995, Planta Med. 61:425-28; Reish, O., etal., 1995, Am. J. Hum. Genet. 57:127-32; Shirota, S., et al., 1994,Biol. Pharm. Bull. 17:266-69; Kameyama, K., et al., 1989,Differentiation 42:28-36). Researchers have also focused on the effectsof intercellular signaling molecules on melanogenesis (see, e.g.,Furumura, M. et al., 1998, Proc. Natl. Acad. Sci. (USA) 95:7374-78;Sakai, C., et al., 1997, EMBO J. 16:3544-52; McLeod, S.D. et al., 1995,J. Endocrinol. 146:439-47).

[0016] Pigmentation disorders of mammals and Drosophila are known and insome cases have been linked to specific defects in proteins involved inintracellular protein trafficking (Spritz (1999) Trends Genet.15:337-340). For example, the hypopigmentation associated withHermansky-Pudlak syndrome (HPS) appears to be related to the regulationof protein trafficking by the AP3 protein . (Dell'Angelica et al. (1999)Mol. Cell 3:11-21).

[0017] For many individuals of all ages, the inappropriate production oroverproduction of melanin is a cosmetic problem. By way of example, manychildren develop freckles after exposure to the sun, and for individualsin middle or advanced age, chloasma, freckles, and pigmentary depositsafter sunburn tend to occur or increase in frequency. In addition, thesepigment deposits do not disappear quickly and are more likely to becomepermanent with advancing age.

[0018] A number of products have been developed to effect a decrease inskin pigmentation. One such product contains hydroquinone, a well knownactive substance for skin de-pigmentation (e.g., see U.S. Pat. No.6,139,854). However, hydroquinone can have serious side effects ifapplied over a long period of time. For example, the application ofhydroquinone to skin may lead to permanent de-pigmentation, and thus toincreased photosensitivity of the skin when exposed to ultravioletlight. For that reason, in some countries hydroquinone is only allowedto be used for skin de-pigmentation in limited concentrations, and, inother countries, the product is banned completely for this application.

[0019] A variety of other substances have been proposed for the controlor inhibition of skin pigmentation. Almost all of these substances workby either bleaching existing pigment or preventing new pigment synthesisby inhibiting the activity of tyrosinase, the principle rate-limitingenzyme in the production of melanin. For example, U.S. Pat. No.6,123,959 describes the use of aqueous compositions comprising liposomesand at least one competitive inhibitor of an enzyme for the synthesis ofmelanin in combination with at least one non-competitive inhibitor of anenzyme for the synthesis of melanin. U.S. Pat. No. 6,132,740 describesthe use of certain resorcinol derivatives as skin lightening agents. WO99/64025A1 describes compositions for skin lightening which containtyrosinase inhibiting extracts from dicotyledonous plant speciesindigenous to Canada. U.S. Pat. No. 5,580,549 describes an externalpreparation for skin lightening comprising 2-hydroxybenzoic acidderivatives and salts thereof as inhibitors of tyrosinase. WO 99/09011A1describes an agent for inhibiting skin erythema and/or skinpigmentation, containing at least one carbostyril derivative and saltsthereof. U.S. Pat. Nos. 5,214,028 and 5,389,611 describes lactoferrinhydrolyzates for use as a tyrosinase inhibitory agents.

[0020] Despite the development of these and other compositions tolighten skin, there remains a need in the art for the development ofless toxic, safer alternatives to skin bleaching and more effective andefficient methods of inhibiting melanin production. The need for new andimproved methods for lightening skin is evident in view of the cosmeticindustry's estimate that the market for skin lighteners worldwideexceeds well over one billion dollars a year. Thus, there is acontinuing need for the development of improved agents that limit orinhibit pigmentation in the skin.

SUMMARY OF THE INVENTION

[0021] The present invention provides novel screens for theidentification of compounds that inhibit or increase melanogenesis inmelanogenic cells. The development of these assays is based, in part, onthe discovery that some compounds that inhibit melanogenesis do so bycausing a mislocalization of tyrosinase, the key enzyme in melaninsynthesis.

[0022] The P protein is a pivotal target for compounds and drugs todecrease or increase pigmentation of the skin, hair and/or eyes.Accordingly, in one aspect, the present invention provides, for thefirst time, screens for compounds that inhibit or enhance P proteinfunction based, in part, on the discovery that P protein function isrequired for proper cellular localization of tyrosinase and othermelanosomal proteins and is required for both full tyrosinase activityand melanogenesis in melanogenic cell types such as, for example,melanocytes and melanoma cells.

[0023] Wild-type melanogenic cells target tyrosinase primarily tomelanosomes. Some tyrosinase is also secreted by these cells. Pprotein-compromised melanogenic cells mislocalize tyrosinase. Theysecrete significantly more tyrosinase than wild-type melanogenic cells,and also contain tyrosinase in non-melanosomal vesicles. Tyrosinase thatis secreted from melanogenic cells, regardless of whether the cells havenormal or inhibited P protein function, is enzymatically active in thegrowth or incubation medium, where it can convert tyrosine into melanin.

[0024] In one aspect, the present invention provides a method ofscreening for compounds that inhibit melanogenesis in melanogenic cells,comprising incubating these cells in medium containing a compound to betested, and identifying compounds that cause a change in the cellularlocalization of tyrosinase in these cells. Mislocalization of tyrosinasecan indicate inhibition of melanogenesis.

[0025] In a still further aspect, the present invention provides methodsof screening for compounds that increase melanogenesis in melanogeniccells, comprising incubating melanogenic cells in medium containing acompound to be tested, and identifying compounds that cause a decreasein the amount of tyrosinase secreted by the cells relative to the amountof tyrosinase retained by the cell, wherein such relative decrease inthe amount of tyrosinase secreted indicates that the compound is acandidate for a compound that increases melanogenesis.

[0026] Another aspect of the invention is based, in part, on thediscovery that non-melanogenic cells can be made to produce activetyrosinase by transfecting them with a heterologous tyrosinase-encodinggene. The tyrosinase activity of these cells is dramatically increasedby cotransfection with a heterologous P protein-encoding gene. Maximaltyrosinase activity in these cells is, therefore, dependent upon Pprotein function. When cells expressing both heterologous tyrosinase andheterologous P protein are treated with drugs that inhibit P proteinfunction such as, for example, imipramine, the tyrosinase activity ofthese cells is reduced to that of cells expressing heterologoustyrosinase alone. Imipramine and other drugs that inhibit P proteinfunction do not otherwise affect tyrosinase activity in cells thatexpress heterologous tyrosinase but that do not express heterologous Pprotein.

[0027] Accordingly, in a further aspect, the present invention providesmethods of screening for compounds that affect (e.g., either inhibit orincrease) P protein function in cells that do not ordinarily expresstyrosinase and/or P protein, comprising manipulating these cells so thatthey express both tyrosinase and P protein, and treating the cells witha compound to be tested. The tyrosinase activity of these cells ismeasured. Compounds that affect (e.g., inhibit or increase) thetyrosinase activity of these cells, but that do not affect thetyrosinase activity of cells expressing tyrosinase alone, are identifiedas compounds that affect tyrosinase in a P protein dependent manner.

[0028] In a further aspect, the present invention provides methods formodeling chemical compounds known to affect or mimic the function of Pprotein. Analogs of the modeled compound are selected or designed, andscreened for the ability to affect or mimic P protein function. By usinganalogs of a compound known to affect or mimic P protein function, newand better compounds that affect or mimic P protein function can bediscovered using the methods of the present invention.

[0029] In a still further aspect, the present invention provides methodsfor using, in medicinal and cosmetic compositions, compounds that affector mimic the function of P protein, thereby treating a disease,condition, or disorder involving the production (underproduction oroverproduction) of melanin.

[0030] The inventors have also determined that agents capable ofmodifying late endosomal/lysosomal transport modify pigmentation byaltering the trafficking of tyrosinase and other proteins necessary formelanin synthesis. This discovery has been utilized to provide otheraspects of the present invention, which include methods andpharmaceutical compositions comprising agents that modify lateendosomal/lysosomal trafficking. These methods and pharmaceuticalcompositions are useful for decreasing and/or inhibiting melaninproduction and, therefore, decreasing, lowering or preventing theformation of skin pigmentation.

[0031] The invention thus further provides, in another aspect, a methodof decreasing, lowering or preventing melanin production in amelanocyte, comprising contacting the melanocyte with a pharmaceuticallyeffective amount of a compound that effects an alteration in lateendosomal/lysosomal trafficking in the melanocyte wherein the alterationin late endosomal/lysosomal trafficking results in a decrease in melaninproduction in the melanocyte. In a certain embodiment, the alteration inlate endosomal/lysosomal trafficking is effected by contacting themelanocyte with a compound that is an antagonist of lateendosomal/lysosomal trafficking. In another certain embodiment, thealteration in late endosomal/lysosomal trafficking is an alteration inlate endosomal/lysosomal cholesterol trafficking.

[0032] In certain embodiments, an alteration in late endosomal/lysosomaltrafficking is effected by contacting the melanocyte with a compoundselected from the group consisting of progesterone, a hydrophobic amine,sphingosine, and a compound of the formula

[0033] wherein X is O or S. In a preferred embodiment, X is O.

[0034] R₁ is —C(O)(C₁-C₆)alkyl or —(CH₂)_(n)—O—(C₁-C₆)alkyl, or—(CH₂)_(n) —NR₇R₈ where n is 0-3, where each of R₇ and R₈ areindependently selected from H and (C₁-C₆)alkyl. Preferably, each of R₇and R₈ are independently selected from —C(O)(C₁-C₃)alkyl,—CH₂—O—(C₁-C₃)alkyl, or —(CH₂)₂—N(C₁-C₃ alkyl)₂. More preferably, eachof R₇ and R₈ is independently selected from —C(O)CH₃, —CH₂—O—CH₃, or—(CH₂)₂—N(CH₃)₂.

[0035] R₂ is H or (C₁-C₆)alkyl. In some embodiments, R₂ is (C₁-C₃)alkyl.In preferred embodiments, R₂ is —CH₃.

[0036] R₃ is H or (C₁-C₆)alkyl. In preferred embodiments, R₃ is(C₁-C₃)alkyl. In more preferred embodiments, R₃ is —CH₃.

[0037] R₄ is —C(O)(C₁-C₆)alkyl. Preferably, R₄ is —C(O)(C₁-C₃)alkyl. Inmore preferred embodiments, R₄ is —C(O)CH₃ or —C(O)CH₂CH₃.

[0038] R₅ is H or —(C₁-C₆)alkyl. Preferably, R₅ is H or —CH₃. In anotherembodiment, R₄ and R₅ taken together are=O.

[0039] R₆ is H or —(C₁-C₆)alkyl or —(CH₂)_(n)—NR₉R₁₀, where each of R₉and R₁₀ are independently selected from H and (C₁-C₆)alkyl. Preferably,R₆ is H or —CH₃, or —CH₂CH₃ or —CH₂NH₂. In another embodiment, R₅ and R₆are combined with the carbon atoms to which they are attached to form aC₅-C₈ carbocyclic ring, and preferably a C₆ carbocyclic ring, which canbe substituted by one to three of halogen, OH, —(C₁-C₆)alkyl,—(C₁-C₆)alkoxy, amino, ═O, (C₁-C₆)alkylamino, di- ((C₁-C₆)alkyl)amino,trifluoromethyl, or —OCF₃, which substituents can be substitutedanywhere on the carbocyclic ring where it is possible to make suchsubstitutions.

[0040] In certain embodiments, the compound is progesterone. In otherembodiments, the compound is a hydrophobic amine. In particularembodiments, the hydrophobic amine is selected from the group consistingof a phenothiazine and a tricyclic antidepressant. In other embodiments,the compound is a phenothiazine. In certain embodiments of theinvention, the phenothiazine is selected from the group consisting oftrifluoperazine, chlorpromazine, prochlorperazine, triflupromazine,promazine, thioridazine, mesoridaine, piperacetazine, perphenazine,fluphenazine, acetophenazine, and thiethylperazine. In yet otherembodiments, the compound is a tricyclic antidepressant. In certainembodiments, the tricyclic antidepressant is selected from the groupconsisting of imipramine, nortriptyline, protriptyline, trimipramine,and doxepin. In other particular embodiments, the compound issphingosine. In another embodiment, the tricyclic antidepressant is notimipramine.

[0041] In still other embodiments, the compound is an antagonist of alate endosomal/lysosomal trafficking protein. In certain embodiments,the compound is of the formula

[0042] wherein the X and R₁-R₆ groups are as defined above. In apreferred embodiment, the compound is selected from the group consistingof:

[0043] and any pharmaceutically acceptable salts or solvates thereof.

[0044] In another aspect, the invention provides a method of reducingskin pigmentation. In this method the skin of a patient in need of saidtreatment is contacted with a pharmaceutically effective amount of acompound that effects an alteration in late endosomal/lysosomaltrafficking, wherein such an alteration results in a reduction of skinpigmentation. In a certain embodiment, the alteration in lateendosomal/lysosomal trafficking is effected by contacting the skin witha compound that is an antagonist of late endosomal/lysosomaltrafficking. In another certain embodiment, the alteration in lateendosomal/lysosomal trafficking is an alteration in lateendosomal/lysosomal cholesterol trafficking.

[0045] In certain embodiments, an alteration in late endosomal/lysosomaltrafficking is effected by contacting the skin with a pharmaceuticallyeffective amount of a compound selected from the group consisting ofprogesterone, a hydrophobic amine, sphingosine, and a compound of any ofthe formulae (I) through (VIII) as defined above, or a pharmaceuticallyacceptable salt or solvate thereof.

[0046] In certain embodiments, the compound is progesterone. In otherembodiments, the compound is a hydrophobic amine. In particularembodiments, the hydrophobic amine is selected from the group consistingof a phenothiazine and a tricyclic antidepressant. In other embodiments,the compound is a phenothiazine. In particular embodiments, thephenothiazine is selected from the group consisting of trifluoperazine,chlorpromazine, prochlorperazine, triflupromazine, promazine,thioridazine, mesoridaine, piperacetazine, perphenazine, fluphenazine,acetophenazine, and thiethylperazine. In yet other embodiments, thecompound is a tricyclic antidepressant. In certain embodiments, thetricyclic antidepressant is selected from the group consisting ofimipramine, nortriptyline, protriptyline, trimipramine, and doxepin. Inanother embodiment, the tricyclic antidepressant is not imipramine. Insome particular embodiments, the compound is sphingosine. In certainembodiments, the compound is an antagonist of a late endosomal/lysosomaltrafficking protein. In certain embodiments, the compound has theformula of any of (I) through (VIII) as defined above, or apharmaceutically acceptable salt or solvate thereof.

[0047] In yet another aspect, the invention provides a pharmaceuticalcomposition for reducing skin pigmentation. This composition comprises apharmaceutically effective amount of a compound that effects analteration in late endosomal/lysosomal trafficking in a skin cell, andfurther comprises a pharmaceutically acceptable carrier. In a certainembodiment, the alteration in late endosomal/lysosomal trafficking iseffected by contacting the melanocyte with a compound that is anantagonist of late endosomal/lysosomal trafficking. In another certainembodiment, the alteration in late endosomal/lysosomal trafficking is analteration in late endosomal/lysosomal cholesterol trafficking. Thepharmaceutical composition is preferably adapted for topicaladministration.

[0048] In certain embodiments, the compound that effects an alterationin late endosomal/lysosomal trafficking is selected from the groupconsisting of progesterone, a hydrophobic amine, sphingosine, and acompound of any of formulae (I) through (VIII) as defined above, or apharmaceutically acceptable salt or solvate thereof.

[0049] In certain embodiments, the compound is progesterone. In otherembodiments, the compound is a hydrophobic amine. In particularembodiments, the hydrophobic amine is selected from the group consistingof a phenothiazine and a tricyclic antidepressant. In some embodiments,the compound is a phenothiazine. In certain embodiments, thephenothiazine is selected from the group consisting of trifluoperazine,chlorpromazine, prochlorperazine, triflupromazine, promazine,thioridazine, mesoridaine, piperacetazine, perphenazine, fluphenazine,acetophenazine, and thiethylperazine. In other embodiments, the compoundis a tricyclic antidepressant. In particular embodiments, the tricyclicantidepressant is selected from the group consisting of imipramine,nortriptyline, protriptyline, trimipramine, and doxepin. In anotherembodiment the tricyclic antidepressant is not imipramine. In otherembodiments, the compound is sphingosine. In certain embodiments, thecompound is an antagonist of a late endosomal/lysosomal traffickingprotein. In certain embodiments, the compound has the structure of anyof formulae (I) through (VIII) as defined above, or a pharmaceuticallyacceptable salt or solvate thereof.

[0050] In another aspect, the invention provides a method of activatingmelanogenesis. The method comprises contacting a melanocyte withdiminished or absent P protein activity with a pharmaceuticallyeffective amount of a compound that inhibits ATPases, whereby theinhibition of ATPases results in an activation of melanogenesis in themelanocyte. In a certain embodiment, the melanocyte with diminished orabsent P protein activity is contacted with a pharmaceutically effectiveamount of a compound selected from the group consisting of bafilomycin,concanamycin, and derivatives thereof. In a preferred embodiment, themelanocyte with diminished or absent P protein activity is contactedwith a pharmaceutically effective amount of bafilomycin, or a derivativethereof. In another preferred embodiment, the melanocyte with diminishedor absent P protein activity is contacted with a pharmaceuticallyeffective amount of concanamycin, or a derivative thereof.

[0051] In another aspect, the invention provides a method of treatingtyrosinase-positive, oculocutaneous albinism in an individual. Themethod comprises contacting skin of the individual with apharmaceutically effective amount of a compound that inhibits ATPases,whereby the inhibition of ATPases results in an activation ofmelanogenesis in the tyrosinase-positive, oculocutaneous albinisticindividual. In a certain embodiment, the skin of the individual iscontacted with a pharmaceutically effective amount of a compoundselected from the group consisting of bafilomycin, or a derivativethereof, and concanamycin, or a derivative thereof. In a preferredembodiment, the skin of the individual is contacted with apharmaceutically effective amount of bafilomycin, or a derivativethereof. In another preferred embodiment, the skin of the individual iscontacted with a pharmaceutically effective amount of concanamycin, or aderivative thereof.

[0052] In another aspect, the present invention further provides a kitcomprising a container comprising a pharmaceutical composition of thepresent invention as described above. The kit may further comprise apackage insert comprising printed instructions directing the use of thepharmaceutical composition for modulating skin pigmentation, e.g., foreither lightening or darkening the skin, as appropriate to theparticular pharmaceutical composition.

BRIEF DESCRIPTION OF THE FIGURES

[0053]FIG. 1 is a graphic presentation of tyrosinase activity in mediafrom cultured melanocytes. Melan-a melanocytes cultured from black mice,and melan-p melanocytes cultured from mice lacking a P gene transcript,were separately cultured in DMEM containing low (0.03 mM) or high (0.3mM) tyrosine for the indicated time period. The activity of tyrosinasewas determined at specific time intervals in media from melanocytes. Themedium was dialyzed prior to determining the enzyme activity, which isexpressed as cpm of tritiated water generated per hour. Δ Melan-a, hightyrosine; ▴ melan-a, low tyrosine; • melan-p1, high tyrosine; °melan-p1, low tyrosine. Increasing tyrosinase activity in the mediaremoved from melan-p cells, which have no P protein transcripts, grownin the presence of 0.03 mM tyrosine, reflects an increased secretion oftyrosinase by these cells. In contrast, melan-a cells, which representwild-type melanocytes, secrete significantly less tyrosinase into themedia. Growing melan-p cells in the presence of high tyrosine partiallyalleviated the P-deficient phenotype.

[0054]FIGS. 2A and 2B are graphic presentations of tyrosinase activityin cell extracts and media from melan-A cells. Cultured melan-amelanocytes were incubated for 48 hours in the presence of benztropine,imipramine, nitroquipazine, or left untreated. Incubation media or cellextracts were assayed for tyrosine hydroxylase activity, as in FIG. 1.Column 1, untreated melanocytes; Column 2, melanocytes treated withbenztropine; column 3, melanocytes treated with10,11-dihydro-n,n-dimethyl-5H-dibenz[b,f]azepine-5-propanamine(imipramine); Column 4, melanocytes treated with6-nitro-2-(1-piperazinyl)-quinoline maleate (nitroquipazine). In FIG. 2A(left), tyrosine hydroxylase activity of melan-a cell extracts ismeasured in cpm [³H]H₂O/60 micrograms protein/hr. In FIG. 2B (right),tyrosine hydroxylase activity in media from melan-a cells is measured incpm [³H]H₂O/hr normalized to the amount of cell extract protein. Thetyrosine hydroxylase activity of extracts from melan-a cells incubatedwith benztropine (column 2 in FIG. 2A.) and nitroquipazine (column 4 inFIG. 2A) is higher than that seen in untreated cells. The extracts fromcells treated with imipramine (column 3 in FIG. 2A) show a reducedactivity. The effects of the drugs on the enzyme activity of the cellextracts is not reflected in the activity of the enzyme secreted intothe media. While benztropine has little effect on activity (column 2 inFIG. 2B), imipramine (column 3 in FIG. 2B) and nitroquipazine (column 4in FIG. 2B) cause a significant increase in activity in the media.

[0055]FIG. 3 is a graphic presentation of relative tyrosinase activityin transfected COS cells. COS cells were transfected with two doses ofthe vector alone (V+V), one dose of the vector alone and one dose of thevector carrying a tyrosinase-encoding gene (V+T), one dose of the vectoralone and one dose of the vector carrying a P protein-encoding gene(V+P), or one dose each of the vectors carrying a tyrosinase-encodinggene and a P protein-encoding gene (T+P). Equal quantities of cellextract protein were assayed for tyrosine hydroxylase activity. Relativeactivities shown are calculated as the activity of the test sampledivided by the activity of the V+T sample. The introduction of anexpression plasmid carrying the tyrosinase gene (V+T) results intyrosine hydroxylase activity in COS cells. This activity is the directresult of the tyrosinase-encoding plasmid, since transfection with theexpression vector (V+V) alone does not generate any tyrosine hydroxylaseactivity. The tyrosine hydroxylase activity in cells carrying thetyrosinase-encoding plasmid can be increased almost 4-fold byco-transfection with the P gene expression plasmid (T+P). This increaseis the result of an interaction between tyrosinase and P protein, sincethe introduction of P (V+P) without tyrosinase generates no tyrosinehydroxylase activity.

[0056]FIG. 4 is a graphic presentation of tyrosinase activity intransfected COS cells. COS cells transfected with a vector carrying atyrosinase-encoding gene, or with a first vector carrying atyrosinase-encoding gene and with a second vector carrying a Pprotein-encoding gene as in FIG. 3, were treated with benztropine,imipramine, nitroquipazine, or left untreated, as in FIG. 2. Cellextracts were prepared as in FIG. 3 The tyrosine hydroxylase activity ofcell extracts was determined as in FIG. 1 as a measure of tyrosinaseactivity. Column 1, untreated transfectants; Column 2, transfectantstreated with benztropine; Column 3, transfectants treated withimipramine; Column 4, transfectants treated with nitroquipazine.Tyrosine hydroxylase activity is measured in cpm [³H]H₂O/60 microgramsprotein/hr. Cells co-transfected with a tyrosinase-encoding gene and a Pprotein-encoding gene (T+P) show a higher tyrosine hydroxylase activitythan cells transfected with a tyrosinase-encoding gene alone (V+T)(column 1). This effect is not altered by incubation of cells in thepresence of benztropine (column 2) or nitroquipazine (column 4). Thepresence of imipramine, however, abolishes the effect of P protein whileappearing to have little effect on the activity in the cells withtyrosinase alone (column 3).

[0057]FIGS. 5A and 5B are graphic presentations demonstrating that thelevels of secreted tyrosinase are elevated in melan-p1 and are reducedby inhibition of cysteinyl proteases. (A) Melan-p1 cells incubated inlow (0.03 mM) tyrosine and high (0.3 mM) tyrosine (TYR) were treated for48 hours with increasing concentrations of the protease inhibitor E64(μM). The tyrosinase activity in the media is expressed as a percentageof total activity in the extract and medium. (B) The concentration ofmelanin was determined by solubilizing the cell pellet and measuring theabsorbance at 470 nm.

[0058]FIGS. 6A and 6B are representations of electronic micrographsdemonstrating the ultrastructure of cultured melanocytes. Theperi-nuclear area of melan-a (A) and melan-p1 (B) melanocytesdemonstrate the Golgi apparatus (G). Melanosomes in the melan-a cell areof stage I, II, III, and predominantly stage IV (arrows). Melanosomes inmelan-p1 cells are predominantly stage I and II with an occasional earlystage III (arrowheads); no stage IV melanosomes were observed. BAR=1.0micron.

[0059]FIGS. 7A and 7B are representations of electronic micrographsdemonstrating ultrastructure of cultured melanocytes processed for DOPAhistochemistry. The perinuclear area of melan-a (A) and melan-p1 (B)melanocytes demonstrating the Golgi apparatus with DOPA reaction productin the cisternae and 50 nm vesicles of the TGN (G). The 50 nm vesiclesare confined to the TGN in the melan-a cells and radiate away from theTGN in melan-p1 cells (arrowheads), and can be observed in closeproximity to the plasma membrane (inset). Occasional stage IIImelanosomes are noted (arrows). BAR=1.0 micron.

[0060]FIGS. 8A and 8B are graphic presentations demonstrating acidphosphatase targeting in melan-A and melan-P cells. Acid phosphataseactivity was measured in fractionated membranes from melan-a (squares)and melan-p cells (circles) as described below in Example 5. FIG.8A=small granule fractions. FIG. 8B=large granule fractions.

[0061]FIGS. 9A and 9B are graphic presentations demonstratingβ-galactosidase targeting in melan-A and melan-P cells. β-galactosidaseactivity was measured in fractionated membranes from melan-a (squares)and melan-p cells (circles) as described below in Example 5. FIG.9A=small granule fractions. FIG. 9B=large granule fractions.

[0062]FIGS. 10A and 10B are graphic presentations demonstratingβ-hexosaminidase targeting in melan-A and melan-P cells.β-hexosaminidase activity was measured in fractionated membranes frommelan-a (squares) and melan-p cells (circles) as described below inExample 5. FIG. 10A=small granule fractions. FIG. 10B=large granulefractions.

[0063]FIGS. 11A and 11B are graphic presentations demonstratingβ-glucosidase targeting in melan-A and melan-P cells. β-glucosidaseactivity was measured in fractionated membranes from melan-a (squares)and melan-p cells (circles) as described below in Example 5. FIG.11A=small granule fractions. Figure 11B=large granule fractions.

[0064]FIGS. 12A and 12B are graphic presentations demonstratingβ-glucuronidase targeting in melan-A and melan-P cells. β-glucuronidaseactivity was measured in fractionated membranes from melan-a (squares)and melan-p cells (circles) as described below in the Example 5. FIG.12A=small granule fractions. FIG. 12B=large granule fractions.

[0065]FIG. 13 is a graphic representation providing melanin opticaldensity (OD) determinations for melan-a melanocytes treated withprogesterone. Cells were treated with no drug, i.e., control, 300 μM1-phenyl-2-thiourea (PTU), 100 μM isobutylmethylxanthine (IBMX), aphosphodiesterase inhibitor, or 20 μM progesterone.

[0066]FIG. 14 is a graphic representation providing melanin opticaldensity (OD) determinations for melan-a melanocytes treated withimipramine. Control cells received no drug treatment, and test cellswere treated with 300 μM PTU, 100 μM IBMX, or 20 μM imipramine (IMP).

[0067]FIG. 15 is a graphic representation providing melanin opticaldensity (OD) determinations for melan-a melanocytes incubated with3β-(2-diethylaminoethoxy)-androstenone HCI (hereinafter “U18666A”).Control cells received no drug treatment, and test cells were treatedwith 300 μM PTU, 100 μM IBMX, 2.5×10⁻⁴ μM U18666A, 2.5×10⁻³ μM U18666A,0.025 μM U18666A, 0.25 μM U18666A, or 2.5 pM U18666A.

[0068]FIG. 16 is a graphic representation providing melanin opticaldensity (OD) determinations for melan-a melanocytes incubated withderivatives of U18666A. Control cells received no drug treatment, andtest cells were treated with 300 μM PTU (Sigma, St. Louis, Mo.), 100 μMIBMX (Sigma, St. Louis, Mo.), 2.5 μM U18666A, and 5 μM U18666A analogs:5 μM CP-59875501, 5 μM CP-602367, 5 μM UK-204039, 5 μM UK-204041, and 5μM UK-204042 (Pfizer, Inc., Groton, Conn.).

[0069]FIG. 17 is a graphic representation providing melanin opticaldensity (OD) determinations for melan-a melanocytes cells treated withU18666A in combination with IBMX. Control cells received no drugtreatment, and test cells were treated with 300 μM PTU, 10 μM IBMX, 2.5μM U18666A, or 10 μM IBMX combined with 2.5 μM U18666A.

[0070]FIG. 18A is a graphic representation providing measurements oftyrosinase activity of extract from melan-a melanocytes incubated withU18666A. Control cells received no drug treatment, and test cells weretreated with 300 μM PTU, 10 μM IBMX, or 2.5 pM U18666A.

[0071]FIG. 18B is a representation of a Western blot in which the amountof tyrosinase protein in melan-a melanocytes treated with U18666A andcontrol untreated cells are compared.

[0072]FIG. 19 is a graphic representation providing measurements oftyrosinase activity on the small granule fractions of a stepwise sucrosegradient fractionation of melan-a melanocytes treated with PTU andU18666A.

[0073]FIG. 20 is a graphic representation providing measurements oftyrosinase activity on the large granule fractions of a stepwise sucrosegradient fractionation of melan-a melanocytes treated with PTU andU18666A.

[0074]FIG. 21 is a graphic representation of the effects of bafilomycinA1 treatment on melanin content in melan-a, melan-p, B16 and melan-p(P10) cells at various concentrations (0.0 μM, 1.0 μM, 2.0 μM, and 2.5μM). Melan-a cells are wild-type melanocytes; melan-p cells aremelanocytes with diminished or absent P protein activity; B16 cells aremelanoma cells that lack tyrosinase and P protein; B16F10 cells aremelanocytes that have detectable levels of tyrosinase, but not Pprotein; and melan-p (P10) cells are melanocytes with diminished orabsent P protein activity stably transfected with an expression plasmidcarrying the pink-eyed dilution gene.

[0075]FIG. 22 is a graphic representation of the effects of concanamycinA treatment on melanin content in melan-a, melan-p, B16 and melan-p(P10) cells at various concentrations (0.0 μM, 0.5 μM, and 1.0 μM).

DETAILED DESCRIPTION OF THE INVENTION

[0076] The patent and scientific literature referred to hereinestablishes knowledge that is available to those with skill in the art.The issued patents, applications, and references that are cited hereinare hereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.

[0077] The invention is based, in part, on the discovery that compoundsthat cause melanogenic cells to mislocalize tyrosinase (e.g., toincrease the amount of tyrosinase secreted or the amount of tyrosinasefound in non-melanosomal vesicles) also inhibit melanogenesis. Forpurposes of the present invention, the term “melanogenic cells” isdefined as cells that contain pigmented melanosomes (e.g., melanocytecells and melanoma cells). Melanogenic cells can include, for example,melanogenic cells that express heterologous melanosomal proteins. Forexample, in preferred embodiments, the coding sequence or sequences ofthe P protein gene, tyrosinase gene, TRP-1 gene, and/or TRP-2 gene in amouse melanogenic cell can be mutated or deleted, and the cellengineered to express instead the corresponding coding sequence of thehuman P protein gene, tyrosinase gene, TRP-1 gene, and/or TRP-2 gene.

[0078] The present invention also relates to the discovery that the Pprotein is necessary to correctly localize tyrosinase predominantly tothe membrane of melanosomes. Yet another aspect of the present inventionis based on the finding that melanocytes treated with compounds thatinhibit P protein function accumulate reduced amounts of intracellularmelanin, and secrete increased amounts of tyrosinase into the growthmedium. Still another aspect of the present invention relates to thediscovery that, in the presence of the P protein, the enzymatic activityof tyrosinase protein in cultured cells is augmented. In yet anotheraspect, the present invention relates to the discovery that whenmelanocytes are contacted with inhibitors of late endosomal/lysosomaltrafficking, thereby affecting tyrosinase localization, melanogenesis isinhibited and melanin synthesis is decreased. Accordingly, the presentinvention provides novel methods of screening for compounds that inhibitmelanogenesis. Compounds identified using the methods of the presentinvention are useful for treating diseases and cosmetic defectsassociated with the underproduction or overproduction of melanin.

[0079] Also disclosed by the present invention is the fact thatwild-type melanogenic cells with normal P protein function secrete sometyrosinase, and that compounds that increase secretion of tyrosinase ina P protein dependent manner also inhibit melanogenesis. Accordingly,the present invention provides novel methods of screening for compoundsthat increase melanogenesis by increasing the function of P protein. Forpurposes of this application, compounds that increase the function of Pprotein and compounds that decrease the function of P protein arecollectively referred to herein as “compounds that affect the functionof P protein.” Still another aspect of the invention is a method ofscreening for compounds that increase melanogenesis by mimicking thefunction of P protein. For purposes of the invention, “compounds thatmimic the function of P protein” are compounds that are not P proteins,yet when they are administered to, or incubated with, melanogenic cellsthat do not contain P protein, they serve to restore at least in partthe correct targeting of tyrosinase to the melanogenic membrane.Melanogenic cells that do not contain P protein may be cells that do notexpress P protein transcripts (such as melan-p cells, described herein)or melanogenic cells that do not express a functional P protein geneproduct.

[0080] It has also been determined that the inhibition of lateendosomal/lysosomal trafficking in melanocytes results in a decrease orinhibition of melanogenesis, and the subsequent decrease in the amountof melanin in melanocytes results in a reduction in skin pigmentation.Thus, compounds that inhibit late endosomal/lysosomal trafficking havethe affect of decreasing or inhibiting melanogenesis and reducingpigmentation in melanocytes and skin tissue.

[0081] The present invention also relates to the discovery that theinhibition of ATPases in melanocytes results in activation ofmelanogenesis. Thus, compounds that inhibit ATPases have the affect ofactivating melanogenesis and increasing the amount of melanin inmelanocytes and skin tissue. This discovery is exploited for thetreatment of tyrosinase positive oculocutaneous albinism to effect anincrease in pigmentation.

[0082] Non-limiting examples of compounds or agents useful in thecompositions and methods of the present invention include compounds thataffect P protein function, e.g., imipramine; compounds that affect lateendosomal/lysosomal trafficking, e.g., progesterone; hydrophobic amines;sphingosine; compounds that are in the same class or are derived fromthe compound U18666A; and compounds that inhibit ATPase activity, e.g.,bafilomycin and concanamycin.

[0083] 1 Methods of Screening for Inhibitors or Inducers ofMelanogenesis

[0084] 1.1 Methods of Screening for Inhibitors of Melanogenesis UsingMelanogenic Cells

[0085] In order for melanogenic cells to engage in robust melanogenesis,they must target their tyrosinase predominantly to the melanosomalmembrane. Consequently, in one aspect, the methods of the presentinvention entail screening for compounds that cause melanogenic cells tomislocalize tyrosinase. P protein function is necessary for the correctcellular localization of tyrosinase.

[0086] Therefore, in another aspect, the methods of the presentinvention entail screening for compounds that inhibit P proteinfunction, thereby causing melanogenic cells to mislocalize tyrosinase.Such methods are based, in part, on the discovery that culturedmelanocytes that have been genetically altered to eliminate P proteinfunction secrete significantly more tyrosinase into the growth mediumthan wild-type melanocytes.

[0087] Compounds such as, e.g., imipramine, that reduce or eliminate Pprotein function will have the desired effect. Thus, the cellularmislocalization of tyrosinase by cells treated with a test compoundindicates that the test compound inhibits melanogenesis. Mislocalizationof tyrosinase resulting in secretion can be detected initially byassaying either the level of tyrosinase activity in the medium or cells,or the level of tyrosinase protein in the medium or cells. Testcompounds that cause an increase in secretion of tyrosinase, or adecrease in intracellular tyrosinase, are candidates for compounds thatinhibit melanogenesis by inhibiting P protein function. Such candidatecompounds can be further investigated for their effect on melanogenesis,and/or for their effects in both the presence and absence of P protein,as described more fully below. If the effect of the candidate compounddepends upon the presence of P protein, then the compound inhibits thefunction of P protein.

[0088] Because growing P-protein-deficient melanocytes in the presenceof high levels of tyrosinase in the medium can partially rescue theP-protein-deficiency, it is preferable, but not necessary, that a screenfor inhibitors of melanogenesis is carried out in the presence of lowamounts of tyrosine in the media, e.g., 0.01-0.05 μm tyrosine, morepreferably 0.014-0.03 μm tyrosine.

[0089] 1.2 Methods of Screening for Inhibitors of Melanogenesis UsingAssays for Tyrosinase Activity

[0090] Wild-type melanogenic cells grown in vitro culture willsynthesize melanin inside of melanosomes as they do in vivo. In thesecultured cells, tyrosinase is found predominantly in the melanosomalmembrane, although some tyrosinase is also secreted. The tyrosinase thatis found in the melanosomal membrane is held in place by a C-terminaltransmembrane domain and has its active site disposed toward themelanosomal lumen. By contrast, in melanogenic cells inhibited formelanogenesis through either a mutation in P protein or a compound thatinhibits P protein function, tyrosinase will be mislocalized. Asignificantly greater fraction of the cells'tyrosinase is secreted fromthe cells into the growth or incubation medium. Additionally, thesecreted tyrosinase polypeptide will be shorter than that found inwild-type cells because it lacks its C-terminal membrane anchor. Thesecreted tyrosinase, however, is enzymatically active in the growth orincubation medium where it can synthesize melanin from extracellulartyrosine. Consequently, tyrosine-containing growth or incubation mediafrom melanogenic cells that have been inhibited for melanogenesis willturn dark. The higher the concentration of tyrosine in the medium, thedarker the medium becomes, and the higher the concentration oftyrosinase in the medium, the faster the medium darkens. Becausemelanogenic cells that are not inhibited for melanogenesis secretesignificantly less tyrosinase, the tyrosine-containing growth orincubation media in which they are cultured will not become as dark.

[0091] This discovery can be used in a novel method of screening toidentify compounds that inhibit or modulate melanogenesis. Melanogeniccells are grown in culture or incubated in medium containing tyrosine.The cells are treated with a test compound. If the test compound causestyrosinase to be mislocalized and secreted from the treated cells, thentyrosine in the medium will be converted into melanin, darkening themedium. An assay is used wherein the color of the medium is compared tothe color of the medium of the melanogenic cells grown or incubatedunder similar conditions but without the test compound (a controlmedium). If the medium of the cells treated with the test compound turnsdarker than the control medium, then the test compound is identified ascandidate for a compound that inhibits melanogenesis.

[0092] More typically, in order to obtain at least semi-quantitativedata, the media from the cells is first filtered, centrifuged and/ordialyzed prior to assay for tyrosinase activity. These types oftreatments remove potentially confounding factors such as cells orparticulate matter (e.g., melanosome or shed membranes) containingtyrosinase that could compete for substrate, and/or remove excess freetyrosine that might compete with labeled substrate. Any of a number ofalternative ways of measuring tyrosinase can be carried out, such as byusing any of the enzymatic tyrosinase activities including, but notlimited to, converting tyrosine to dihydroxyphenylalanine (DOPA), DOPAto dopaquinone, and oxidation of 5,6-dihydroxyindole to5,6-indolequinone. For example, when assaying for the tyrosinehydroxylase activity of tyrosinase, non-tyrosine or altered tyrosinesubstrates of tyrosinase can be used in addition to tyrosine. In oneaspect, melanogenic cells are grown or incubated in culture with a testcompound. After pretreatment of the medium, a non-tyrosine or alteredtyrosine substrate of tyrosinase is added to the growth or incubationmedium. The substrate can be a homolog, analog, or derivative oftyrosine which can be a natural product or produced synthetically.Tyrosinase activity in the medium converts the substrate into itsproduct.

[0093] An assay is then used to detect the presence of the productand/or the absence of the substrate. One non-limiting assay is acalorimetric assay. In methods of screening for compounds that inhibitmelanogenesis that use a calorimetric assay, a substrate is chosen thatchanges color when it is acted upon by tyrosinase. That is, thewavelength of light absorbed by the substrate is different than thewavelength of light absorbed by the products of the reaction catalyzedby tyrosinase. The wavelength of light absorbed by the substrate and/orby the products can be in the visible light range, the infrared range,or the ultraviolet range of the spectrum. The concentration ofsubstrate, incubation time, and other reaction conditions can be chosensuch that the speed and/or intensity of the color change is proportionalto the amount of tyrosinase activity in the cells growth or incubationmedium. The color change can be detected by direct observation, ormeasured by a device, such as a spectrophotometer and compared, e.g., toa standard curve prepared using varying amounts of product.

[0094] An example of a colorimetric assay is the DOPA oxidase assay. Inone method of screening for compounds that inhibit melanogenesis usingthis assay, a compound to be tested for its ability to inhibitmelanogenesis is added to the growth or incubation medium of melanogeniccells. After filtration, centrifugation and/or dialyzation of themedium, L-DOPA is added under conditions that would otherwise allow fortyrosinase to catalyze the formation of dopachrome from L-DOPA. In apreferred though non-limiting embodiment, the final concentration ofL-DOPA in the medium is about 5×10⁻³ M, the pH is about 7.4, and thetemperature is about 25° C. Increased absorbance of the medium at 475 nm(relative to the absorbance at 475 nm of medium from similar cells grownunder similar conditions but without the test compound) indicates theformation of dopachrome by tyrosinase in the medium and, therefore, theinhibition of melanogenesis by the test compound.

[0095] Alternatively, as dopachrome absorbs light within the visiblerange, the presence of dopachrome, and hence the inhibition ofmelanogenesis, can be determined by direct inspection of the reaction,without the aid of a spectrophotometer.

[0096] Another assay is a radiometric assay. In an alternative method ofscreening for compounds that inhibit melanogenesis using this assay,substrate is radioactively labeled and added to the growth or incubationmedium to be assayed. If tyrosinase is present in the medium, it cleavesthe substrate into a labeled product and an unlabeled product. Theamount of radioactive substrate that has been converted into radioactiveproduct is measured. The concentration of substrate, time of incubation,temperature of incubation, and other reaction conditions can be chosenso that the amount of radioactive product produced is proportional tothe amount of tyrosinase in the growth or incubation medium beingassayed. A greater amount of labeled product in the medium from cellstreated with the test compound than in the medium of similar cells grownunder similar conditions but without the test compound indicates thatthe test compound is a candidate for a compound that inhibitsmelanogenesis.

[0097] An example of this type of assay is the radiometric tyrosinehydroxylase assay. In this assay, the amount of [³H]H₂O released from[³H]tyrosine as a result of the tyrosine hydroxylase activity of thetyrosinase enzyme is measured. In one method of screening for compoundsthat inhibit melanogenesis that uses this assay, media from melanogeniccells is harvested and cells removed. Additionally, the media can bedialyzed before assay. For assays, 1.5 μCi [³H]tyrosine is added to themedia and incubated for defined lengths of time at appropriatetemperature for enzyme activity. Unreacted [³H]tyrosine is removed fromthe medium by adsorption onto 10% (w/v) activated charcoal in 0.1 Mcitric acid, then treated with 50% (w/v) Dowex resin solution. Themedium is mixed with scintillant and counted in a beta-counter. Asignificant increase in [³H]H₂O levels in the medium of cells that weretreated with a test compound compared to [³H]H₂O levels in the medium ofsimilar cells grown under similar conditions without test compoundindicates that the test compound is a candidate for a compound thatinhibits melanogenesis.

[0098] Yet another example of this type of assay is the radiometricmelanin synthesis assay. In this assay, the amount of [¹⁴C]tyrosine or[¹⁴C]DOPA incorporated into [¹⁴C]melanin is measured. In a non-limitingexample of a method of screening for compounds that inhibitmelanogenesis that uses this assay, melanogenic cells are grown orincubated in medium containing a test compound. The medium is harvestedand 1 μCi [¹⁴C]tyrosine is added and incubated at the appropriatetemperature for four hours. The reaction is terminated with ice-cold 10%(w/v) TCA and the mixture vortexed and frozen for 24 hours. The mixtureis then thawed and centrifuged at 1000 g for 15 minutes at 4° C. Thepellet is resuspended in ice-cold 5% TCA (w/v). This step is repeatedtwice. The final pellet containing [¹⁴C]melanin is solubilized inSoluene®-350 (Packard Instrument Company, Meriden, Conn.) for fourhours, mixed with scintillant, and counted. Alternatively, the pelletcan be collected on filter paper and counted. A significant increase in[14C]melanin levels in media of cells that were treated with a testcompound compared to [14C]melanin levels in media of similar cells grownunder similar conditions, but without the test compound, indicates thatthe test compound is a candidate for a compound that inhibitsmelanogenesis.

[0099] Another assay is a fluorescence assay. In this assay, thesubstrate and/or its products are fluorescent. The wavelength of lightabsorbed and/or emitted by the substrate is different from thewavelength of light absorbed and/or emitted by the products. In anon-limiting example of a method of screening for compounds that inhibitmelanogenesis that uses this assay, melanogenic cells are grown inculture in the presence or absence of the test compound. After a periodof growth or incubation, the media is removed, tyrosinase substrateadded, and a fluorometer used to measure the fluorescence of the growthor incubation medium. The concentration of substrate, time ofincubation, temperature of incubation and other reaction conditions canbe chosen so that the change in fluorescence is proportional to thelevels of tyrosinase activity in the medium being analyzed. Asignificant difference in fluorescence levels of media from cellstreated with a test compound compared to fluorescence levels of mediafrom similar cells grown under similar conditions but without the testcompound, indicates that the test compound is a candidate for a compoundthat inhibits melanogenesis.

[0100] Another type of assay involves the precipitation of reactionproducts. In an example of a method of screening for compounds thatinhibit melanogenesis that uses this assay, a substrate of tyrosinase isincubated with the harvested growth or incubation medium underconditions that promote the activity of tyrosinase. The substrate isacted upon by tyrosinase to produce a reaction product that can beprecipitated. The reaction product is precipitated. The reaction productcan be precipitated from the medium by, for example, increasing ordecreasing the temperature of the medium, increasing or decreasing thepH of the medium, increasing or decreasing the ionic strength or saltconcentration of the medium, or otherwise altering the mediumappropriately, or by centrifugation if the reaction product isinsoluble. Substrate concentrations, time of incubation, temperature ofincubation, and other reaction conditions can be chosen so that theamount of precipitable reaction product is proportional to the levels oftyrosinase activity in the medium being analyzed. A significant increasein the amount of reaction product precipitated from the media of cellstreated with a test compound compared to the amount of reaction productprecipitated from the media of similar cells grown under similarconditions but without the test compound indicates that the testcompound is a candidate for a compound that inhibits melanogenesis.

[0101] 1.3 Methods of Screening for Inhibitors of Melanogenesis UsingAssays for Tyrosinase Protein

[0102] The preceding methods of screening serve to identify inhibitorsof melanogenesis using assays of tyrosinase activity (i.e., theprotein's enzymatic activities). The present invention further providesa method of screening inhibitors of melanogenesis using assays foreither extracellular or intracellular tyrosinase protein levels. Asexplained above, tyrosinase is primarily localized to the melanosomalmembrane in melanogenic cells. Compounds that cause tyrosinase to bemislocalized serve to inhibit melanogenesis. In the following methods ofscreening, assays for determining tyrosinase protein levels and/orlocations are used. This can be done using any of the standardtechniques of protein detection known in the art. The protein detectionassays employed herein can be those described in Harlow and Lane(Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety. These assays include,but are not limited to, immunological assays, including Western blots,solid-phase radioimmunoassays, in situ hybridizations, andimmunoprecipitations. Anti-tyrosinase antibodies are known in the art,and novel anti-tyrosinase antibodies can be generated using well-knowntechniques. Id.

[0103] In a non-limiting method of screening for compounds that inhibitmelanogenesis, melanogenic cells are grown or incubated in mediumcontaining a test compound. The presence, concentration, or amount oftyrosinase in the medium is determined using a protein detection assayas described above. Test compounds that cause treated cells to secretemore tyrosinase than similar cells grown or incubated under similarconditions but without the test compound are candidates for compoundsthat inhibit melanogenesis.

[0104] Another type of assay that can be used in this screen determinesthe cellular localization of tyrosinase protein. In wild-typemelanogenic cells, most tyrosinase is targeted to the melanosomalmembrane, while some tyrosinase is secreted. Mutations or compounds thatinhibit melanogenesis (e.g., mutations or compounds that inhibit Pprotein function) can cause tyrosinase to be secreted to the medium ingreater amounts or to be mislocalized to non-melanosomal vesicles. Thesenon-melanosomal vesicles can be separated from melanosomes usingsubcellular fractionation techniques. In a non-limiting example of amethod of screening for compounds that inhibit melanogenesis that usesthis assay, melanogenic cells are grown or incubated in mediumcontaining a test compound and the cells are harvested. The subcellulardistribution of tyrosinase is then determined in these cells andcompared to the subcellular distribution of tyrosinase in similar cellsgrown or incubated under similar conditions but without the testcompound. The assay can incorporate any technique or combination oftechniques known in the art, including subcellular fractionation (forexample, by sucrose or Percoll density gradient centrifugation), Westernblotting of the cells contents, and tyrosinase activity assays of eachsubcellular fraction. A decrease in the fraction of total tyrosinaseprotein found in the melanosomal fraction, or an increase in thefraction of total tyrosinase protein found in a non-melanosomalfraction, in cells treated with the test compound relative to cells nottreated with the test compound indicates that the test compound inhibitsmelanogenesis.

[0105] Other qualitative assays can be used, such as, e.g., microscopicexamination of cells treated with the test compound. For example, cellstaining techniques, as known in the art, can be used. Cells are grownor incubated in medium containing tyrosine and in the presence of a testcompound. The cells are stained using anti-tyrosinase antibodies, thenexamined microscopically. In a non-limiting example of a method ofscreening using this type of assay, melanogenic cells are grown orincubated in medium containing a test compound, and prepared for cellstaining using techniques commonly known in the art. See, e.g., Harlowand Lane, 1988, supra. Prepared cells are stained using anti-tyrosinaseantibodies. The anti-tyrosinase antibodies can be conjugated to a moietyallowing for its detection. Preferably, a secondary antibody is used.The secondary antibody recognizes and binds to the anti-tyrosinaseantibody. Preferably, the secondary antibody is conjugated to a moietyallowing for its detection. Alternatively, a tertiary antibody can alsobe used. The tertiary antibody is preferably conjugated to a moietyallowing for its detection. Examples of moieties allowing for thedetection of antibodies include fluorescent molecules (for example,fluoroscein, rhodamine, Hoechst 33258, or Texas red), enzymes (forexample, horseradish peroxidase, alkaline phosphatase, orbeta-galactosidase), gold particles, radioactive isotope, and biotin. Anassay is selected based on the labeling moiety used. For example,fluorescence microscopy can be used to detect fluorescently labeledantibodies. For cells stained with enzyme-conjugated antibodies, thecells are further treated with an appropriate substrate for conversionby the antibody-bound enzyme, followed by examination by lightmicroscopy. Gold-particle labeled antibodies can be detected using lightor electron microscopy. Isotope-labeled antibodies can be detected usingradiation-sensitive film. For cells stained with biotin-conjugatedantibodies, the cells are further treated with streptavidin or avidin.The streptavidin or avidin is conjugated to a moiety that allows fordetection such as, for example, a fluorescent molecule, an enzyme, goldparticles, or radioactive isotope. Preferably, the cells are co-stainedwith an antibody or antibodies specific for particular subcellularcompartments (e.g., endosomes, lysosomes, melanosomes, etc.). Using anyone of these techniques, or any other known technique for detectingantibodies in antibody-stained cells, the subcellular distribution oftyrosinase can be determined. If the test compound causes an increasedamount of tyrosinase to be found in non-melanosomal vesicles, and lesstyrosinase in melanosomes, then it inhibits melanogenesis.

[0106] Another type of assay can be used to determine the presence orabsence of the C-terminal portion of the tyrosinase protein. This assaydepends, in part, on the discovery that melanogenic cells inhibited formelanogenesis (e.g., by mutations or compounds that inhibit P proteinfunction) contain and secrete a version of tyrosinase that lacks theC-terminal portion of tyrosinase, including its transmembrane domain andits protein sorting signal. As explained above, this truncated form oftyrosinase nonetheless retains catalytic activity. In a non-limitingexample of a method of screening based on this assay, melanogenic cellsare grown or incubated in the presence of a test compound. An assay isselected that allows the length and/or mass of tyrosinase protein to bedetermined. For example, Western blots or other immunohistochemicaltechniques using antibodies that recognize the N-terminal or centralportions of the tyrosinase protein, or other standard molecularbiological techniques useful for the determination of protein length ormass, can be performed on extracts of these cells and/or on their growthor incubation medium. Antibodies appropriate for these assays can beprepared using standard immunological techniques. See, e.g., Harlow andLane, 1988, supra. If the assay reveals the presence of a shorter orlower molecular weight form of tyrosinase, relative to similar cellsgrown or incubated under similar conditions but without the testcompound, then the test compound inhibits melanogenesis. Alternatively,Western blots or other immunohistochemical techniques using antibodiesrecognizing the C-terminal portion of tyrosinase (e.g., the anti-PEP7antibody prepared as described in Jimenez et al., 1991, J. Biol. Chem.266:1147-1156) can be used in the assay. In these assays, a reduction inthe amount of tyrosinase protein detected by the antibodies indicatesthat the test compound inhibits melanogenesis, because the truncatedtyrosinase lacks the sequences recognized by the antibodies.

[0107] Both full length tyrosinase, and the truncated tyrosinase foundin and secreted by melanogenic cells with inhibited or absent P protein,remain catalytically active when run on non-denaturing polyacrylamidegels. This observation is the basis, in part, of another assay for thetruncated tyrosinase protein. Thus, melanogenic cells can be grown orincubated in medium containing a compound to be tested. Either thegrowth or incubation medium is collected, or cell extracts are prepared,and subjected to non-denaturing polyacrylamide gel electrophoresis.Smaller, more flexible proteins will migrate farther than largerproteins with more complicated three-dimensional structure. Filter paperor a membrane (e.g., nitrocellulose) is soaked in L-DOPA and applied tothe gel. Active tyrosinase in the gel converts L-DOPA into melanin,creating dark spots on the filter or membrane indicating the locationand, therefore, the relative size, of tyrosinase. If cells treated withthe test compound produce two spots on the filter or membrane, whereinone spot indicates tyrosinase of the same size as produced by similarcells grown under similar conditions but without the test compound, andthe other spot indicates tyrosinase of smaller relative size, then thetest compound is a candidate for a compound that inhibits melanogenesis.

[0108] Full length tyrosinase in wild-type melanogenic cells with normalP protein function is found primarily in the insoluble fraction of cellextracts. To be released, it must be solubilized with a detergent (e.g.,Triton X-100™). In contrast, the smaller truncated version of tyrosinasein melanogenic cells with inhibited P protein function is found invesicles in the soluble fraction. These observations are the basis, inpart, of another assay that can be used to detect truncated tyrosinasein P protein-compromised cells. Thus, melanogenic cells are grown orincubated in medium containing a compound to be tested. The cells areharvested and can be subjected to a detergent phase separation toseparate membrane-anchored proteins from soluble proteins. For example,the cells can be solubilized on ice in a buffer containing Triton X-114™detergent. Insoluble contaminants can be spun out at 4° C. Then thesupernatant, which contains solubilized proteins, is phase-separated atroom temperature or elevated temperatures into a detergent phase and anaqueous phase. The ratio of tyrosinase in the detergent phase (whichwill contain tyrosinase proteins containing the C-terminal portion ofthe protein which anchors tyrosinase in the membrane) to tyrosinase inthe aqueous phase (which will contain tyrosinase proteins which lack theC-terminal portion) is determined. Alternatively, cells are harvestedand membranes disrupted by a freeze/thaw cycle or cycles. The disruptedcells are then separated into a soluble fraction and a membrane-bound,insoluble fraction. The ratio of soluble tyrosinase in the solublefraction to insoluble, membrane-bound tyrosinase in the membranefraction is determined. If cells treated with the test compound havehigher levels of soluble tyrosinase than insoluble, membrane-boundtyrosinase than that from similar cells grown under similar conditionsbut without the test compound, then the test compound is a candidate fora compound that inhibits melanogenesis.

[0109] 1.4 Other Methods of Screening for Inhibitors of Melanogenesis

[0110] As described above, the mislocalization and secretion oftyrosinase, and the reduction of tyrosinase activity, are not the onlyresults of an inhibition of melanogenesis. Other melanogenic enzymes arealso affected, as is the biogenesis of melanosomes.

[0111] Inhibition of melanogenesis by mutations that inhibit P proteinfunction can cause a marked alteration in the amount of severalmelanogenic proteins produced in melanocytes, including the TRP-1,TRP-2, and LAMP-1 gene products (Orlow, S. J., and Brilliant, M. H.,1999, supra). In the eyes of wild-type mice, the levels of TRP-1 andTRP-2 gene products are high at birth, fall sharply, increase graduallyto another peak at about 2 weeks, then permanently fall to undetectablelevels by about 40 days. In mice that lack P protein function, forexample, the levels of these proteins are much lower at birth and areundetectable after only a few days (id.).

[0112] Another effect of inhibited melanogenesis caused by a mutationwhich inhibits P protein function is the disruption of a high molecularweight complex comprising tyrosinase, TRP-1 protein, and TRP-2 protein(Orlow, S. J. et al., 1994, supra). For purposes of the presentinvention, the term “high molecular weight complex” is defined as agroup of proteins bound to each other via covalent and/or non-covalentbonds that remain associated with each other during non-denaturing gelfiltration, HPLC, or sucrose gradient sedimentation and have an apparentmolecular weight of between about 200 kD and about 700 kD. In wild-typemelanogenic cells, this “melanogenic complex,” which is associated withthe melanosome, contains a significant fraction of the cells'complementof tyrosinase, TRP-1 protein and TRP-2 protein. In melanogenic cellsinhibited for melanogenesis by inhibition of P protein function, verylittle of any of these proteins is found in high molecular weightcomplexes.

[0113] Another assay takes advantage, in part, of these effects. In anon-limiting example of a method of screening for compounds that inhibitmelanogenesis that uses this type of assay, melanogenic cells are grownor incubated in medium containing a compound to be tested. The cells areharvested, disrupted, and fractionated. This fractionation can be done,for example, using sucrose gradient sedimentation. Aliquots from thesedimentation are assayed for the presence of melanogenic proteins suchas, for example, by using assays for tyrosinase, TRP-1, and/or TRP-2activity, or by using immunohistochemical assays, such asimmunoblotting. Increased amounts of any of these three proteins in lowdensity aliquots and/or decreased amounts of any of these three proteinsin high density aliquots, relative to similar cells grown or incubatedunder similar conditions but without the test compound, indicate thatthe test compound is a candidate for a compound that inhibitsmelanogenesis.

[0114] Another consequence of inhibited melanogenesis can be theaberrant development of melanosomes. Wild-type melanogenic cellstypically contain abundant, fully developed, darkly pigmentedmelanosomes. Such fully developed, darkly pigmented melanosomes are lessabundant or absent in melanocytes inhibited for melanogenesis due to amutation in the P protein-encoding gene when they are grown or incubatedin medium containing low concentrations of tyrosine. Rather, these cellscontain an unusually large number of immature melanosomes. Thisphenomenon is the basis, in part, for another assay that can be used. Ina non-limiting example of a method of screening for compounds thatinhibit melanogenesis that uses this type of assay, melanogenic cellsare grown or incubated in medium containing a test compound. The number,size, shape, and/or color of the melanosomes in the cells is assayed.Such assays are well known in the art. For example, cells can be fixedand stained and examined using light microscopy. Alternatively, cellscan be fixed, stained, sectioned, and examined using electronmicroscopy. Alternatively, cells can be fractionated using densitycentrifugation. Mature melanosomes are denser than immature melanosomes,and so can be separated from them on the basis of density using wellknown techniques. Cells treated with a test compound that havemelanosomes that are altered in number, size, shape, and/or colorcompared to melanosomes from similar cells grown or incubated undersimilar conditions but without the test compound indicates that the testcompound inhibits melanogenesis.

[0115] 1.5 Methods of Screening for Inhibitors of P Protein Function

[0116] As explained above, the P protein plays a pivotal role inmelanogenesis. Melanocytes with loss of function mutations in the Pprotein-encoding gene are inhibited for melanogenesis. In P deficient orP inhibited cells, inhibition of melanogenesis is correlated withmislocalization of tyrosinase. Whereas in wild-type melanocytestyrosinase is localized primarily to melanosomes, in melanocytes withloss of function mutations in the P protein-encoding gene, tyrosinase ispredominantly secreted or found in non-melanosomal vesicles. Inhibitionof melanogenesis and the mislocalization of tyrosinase can be mimickedby treating wild-type melanocytes with compounds that inhibit thefunction of P protein (e.g., imipramine).

[0117] These discoveries are the basis, in part, for a number of screensfor inhibitors of melanogenesis. These screens serve to identifyinhibitors of melanogenesis by identifying inhibitors of P proteinfunction. Thus, melanogenic cells are grown or incubated in mediumcontaining a compound to be tested for its ability to inhibit P proteinfunction. The effect, if any, of the compound can be determined using,for example, any one of the assays described above. In a non-limitingembodiment, the activity of tyrosinase in the growth or incubationmedium of the cells can be measured. For example, tyrosine can be addedto the medium, and its conversion to melanin monitored. Alternatively,non-tyrosine or altered tyrosine substrates of tyrosinase can be addedto the medium, and their conversion into reaction products by tyrosinasecan be followed by, for example, colorimetric assays (e.g., the DOPAoxidase assay), radiometric assays (e.g., the radiometric hydroxylase orradiometric melanin synthesis assays), fluorescence assays, or by theprecipitation of reaction products. These assays are described in detailherein above.

[0118] Alternatively, the assays of tyrosinase protein may be used.These assays can measure, for example, the amount of tyrosinase in thegrowth or incubation medium of the cells treated with the compound to betested, the cellular localization of tyrosinase (e.g., by subcellularfractionation of the cells, or by staining and microscopic examinationof the cells), or the length or mass of the tyrosinase molecules withinthe cells. These assays are described in detail herein above.

[0119] Other assays that can be used include those that measure othereffects of the inhibition of P protein function. For example, theseassays can measure the amount or activity of TRP-1 and/or TRP-2 proteinin cells treated with the compound to be tested, the abundance orcomposition of the high molecular weight melanogenic complex, or thepresence or absence of aberrant melanosomes. These assays are describedin detail herein above.

[0120] Still another assay that can be used involves measuring theintracellular targeting, intracellular levels and/or secretion of acertain class of lysosomal hydrolases. Normally, newly synthesizedlysosomal hydrolases are transported from the trans-Golgi network to alate endosome compartment, portions of which are thought to eventuallyfuse with or form lysosomes. These lysosomes, containing most of theintercellular lysosomal hydrolase activity, can be detected in a largegranule fraction prepared from fractionated cells. As illustrated belowby way of a non-limiting example, some, but not all, lysosomalhydrolases are not correctly targeted to the lysosome-containing largegranule fraction from melan-p cells as opposed to melan-a cells. Inparticular, lysosomal hydrolases that are transported from thetrans-Golgi network to the late endosome via binding to themannose-6-phosphate/insulin-like growth factor type II receptor (the“M6P/IGF-II receptor”) do not accumulate in the large granule fraction.Such incorrectly targeted lysosomal hydrolases include β-hexosaminidase,β-glucosidase, β-glucuronidase and β-galactosidase. In contrast, acidphosphatase, which is not transported to the late endosome via theM6P/IGF-II receptor, correctly accumulates in the large granule fractionin both melan-a and melan-p cells. Thus, P protein function is alsonecessary for the correct targeting of lysosomal enzymes that aretransported to the late endosome via the M6P/IGF-II receptor. Thedefault pathway for such enzymes is secretion into the exterior of thecell.

[0121] These results are the basis, in part, for additional methods ofscreening for compounds that affect P protein function. Accordingly, inlieu of, or in addition to, assays for the mislocalization oftyrosinase, one can screen for the effect of a test compound on thelevel and/or localization of any lysosomal hydrolase that is normallytransported to the late endosome via the M6P/IGF-II receptor including,but not limited to, β-hexosaminidase, β-glucosidase, β-glucuronidase andβ-galactosidase. Since these lysosomal hydrolases are, like tyrosinase,proteins and more particularly enzymes, any of the methods describedabove to assay for the presence of tyrosinase's enzymatic activityand/or protein can be adapted to assay lysosomal hydrolases. Assays forthe enzymatic activity of these enzymes are well known in the art (and,in part, illustrated below by way of a non-limiting example), as aretheir amino acid structures and antibodies that recognize the same. Forexample, one can assay for the presence and/or levels of lysosomalhydrolases in whole cells or cell extracts, in the large granulefraction of a cell extract, and/or in the medium from cells treated withtest compounds. Compounds that cause either a decrease in accumulationof such lysosomal enzymes in cells or, more particularly, the largegranule fraction, or an increase in secretion of such lysosomal enzymes,are candidates for compounds that inhibit the function of P protein;such candidate compounds are then further analyzed using one of theother methods of the invention.

[0122] 1.6 Methods of Screening for Compounds that IncreaseMelanogenesis. Increase P Protein Function and/or Mimic P ProteinFunction

[0123] As explained above, wild-type melanogenic cells typically secretea portion of their tyrosinase into in vitro culture medium. Although thesecreted tyrosinase is enzymatically active, it cannot contribute tomelanogenesis, which occurs inside the cells melanosomes. As describedabove, the level of melanogenesis within melanogenic cells isproportional to the fraction of tyrosinase that is localized tomelanosomes. Compounds that decrease the amount of tyrosinase localizedto melanosomes serve to inhibit melanogenesis. Conversely, compoundsthat reduce the amount of tyrosinase that is secreted, and therebyincrease the amount of tyrosinase localized to melanosomes, are expectedto increase melanogenesis. As explained above, P protein activity isrequired for the localization of tyrosinase to melanosomes. Thus,compounds that increase the activity of P protein in melanogenic cells,as well as compounds that mimic the activity of P protein, will increasemelanogenesis by reducing the amount of tyrosinase that is secreted.

[0124] A number of screens based, in part, on these observations andpredictions can be used to identify compounds that increasemelanogenesis, increase the function of P protein, and/or mimic thefunction of P protein. For example, variations of the assays describedabove using melanogenic cells to identify inhibitors of melanogenesisand P protein function can be used. Melanogenic cells are grown orincubated in vitro in medium containing a compound to be tested for itsability to increase melanogenesis, increase P protein function or mimicP protein function. The effect, if any, of the compound can bedetermined using, for example, any one of the assays described above.For example, the activity of tyrosinase in the growth or incubationmedium of the cells can be measured. A decrease in the activity oftyrosinase in the medium may indicate that less tyrosinase is beingsecreted, and that the compound might therefore increase melanogenesisor P protein function.

[0125] Alternatively, or in addition, melanogenic cells that do notcontain P protein (e.g., melan-p cells) can be used to screen forcompounds that mimic P protein function. In one type of assay that canbe used in the invention, melanogenic cells that do not contain Pprotein are incubated in medium containing a compound to be tested forits ability to mimic P protein function and increase melanogenesis. Incontrast to normal melanogenic cells, such melanogenic cells that do notcontain P protein are light colored in culture (as well as in theanimal). If the melanogenic cells that do not contain P protein turndarker in the presence of the compound than in the absence of thecompound, then the compound mimics P protein function in whole or inpart. The color of the cells can be measured qualitatively such as, forexample, by visual inspection, or quantitatively, such as, for example,by reflectance. Alternatively, melanogenic cells that do not contain Pprotein are treated with the compound to be tested, and the amount oftyrosinase secreted into the medium is assayed. If the amount oftyrosinase in the medium from melanogenic cells that do not contain Pprotein (e.g., melan-p cells) decreases when the cells are treated withthe test compound, then the test compound is a candidate for a compoundthat mimics P protein function. Tyrosinase activity in the medium can bemeasured, for example, by using any of the techniques described above.For example, tyrosine can be added to the medium, and its conversion tomelanin monitored.

[0126] Alternatively, assays of tyrosinase protein may be used. Theseassays can measure, for example, the amount of tyrosinase in the growthor incubation medium of the cells treated with the compound to betested, the cellular localization of tyrosinase (e.g., by subcellularfractionation of the cells, or by staining and microscopic examinationof the cells), or the length or mass of the tyrosinase molecules presentwithin the cells. A decrease in the amount of tyrosinase proteinsecreted into the medium, or an increase of tyrosinase protein inmelanosomes, indicates that the test compound is a candidate for acompound that increases melanogenesis or mimics or enhances P proteinfunction. If the test compound causes similar effects in melanogeniccells that do not contain P protein (e.g., melan-p cells), such a resultwould indicate that the compound mimics P protein function. These assaysare described in detail herein above.

[0127] Other assays that can be used include those that measure othereffects of an increase in P protein function, mimic of P proteinfunction, and/or an increase in melanogenesis. For example, these assayscan measure the amount of TRP-1 and/or TRP-2 protein or activity incells treated with the compound to be tested, the abundance orcomposition of the high molecular weight melanogenic complex, or thepresence or absence of aberrant melanosomes as described above. Anincrease in the amount of TRP-1 and/or TRP-2 protein or activity, anincrease in the amount of these proteins found in high molecular weightmelanogenic complexes, or an increase in the number of high molecularweight complexes, indicates that the test compound is a candidate for acompound that increases melanogenesis or P protein function. If the testcompound causes similar effects in melanogenic cells that do not containP protein (e.g., melan-p cells), such a result would indicate that thecompound mimics P protein function. These assays are described in detailherein above.

[0128] In a variation of these screens, the amount of secretedtyrosinase is compared to the amount of intracellular tyrosinase.Melanogenic cells are grown or incubated in medium containing a compoundto be tested. Using, for example, any of the assays described above, theamount of tyrosinase in the growth or incubation medium is determinedand the amount of tyrosinase within these cells is also determined. Theratio of intracellular tyrosinase to secreted tyrosinase is thencalculated. If this ratio is higher for cells treated with the compoundto be tested than for similar cells grown under similar conditions butwithout the compound, then the compound increases melanogenesis. In anon-limiting preferred embodiment, such a change in the ratio ofintracellular tyrosinase to secreted tyrosinase is observed without achange (e.g., reduction) in the total amount of tyrosinase produced bythe cell. Similarly, if melanogenic cells that do not contain P protein(e.g., melan-p cells) are grown or incubated in medium containing thecompound to be tested, and the ratio of intracellular tyrosinase tosecreted tyrosinase is higher for cells treated with the compound thanfor untreated cells, then the compound can mimic P protein function, andthereby increase melanogenesis.

[0129] 1.7 Methods of Screening for Compounds That Affect P ProteinFunction Using Non-Melanogenic Cells

[0130] Most non-melanogenic cells do not express P protein ortyrosinase. For purposes of the present invention, the term“non-melanogenic cells” is defined as cells that do not containmelanosomes. However, non-melanogenic cells can be made to express bothP protein and tyrosinase, and to synthesize melanin. For purposes of thepresent invention, the term “cells made to express both P protein andtyrosinase” is defined as cells that do not ordinarily express P proteinand/or tyrosinase, but are caused to express both P protein andtyrosinase using any technique known in the art such as, e.g., moleculargenetic techniques. For example, heterologous tyrosinase and/or Pprotein genes can be introduced into the cells by, e.g., transfection,transformation, or transduction. For purposes of the present invention,the term “heterologous” is defined as describing a gene or gene productthat does not naturally exist in that organism, or a gene or geneproduct that is not normally expressed in that cell type. Alternatively,endogenous, but normally quiescent, tyrosinase and/or P protein-encodinggenes can be activated to express tyrosinase and/or P protein (e.g.through targeted homologous recombination of transcriptional controlsequences, or any other activation method). Several methods of thepresent invention are based, in part, on the discovery thatnon-melanogenic cells expressing P protein and tyrosinase together havealmost four times as much tyrosinase activity as cells expressingtyrosinase alone. Cells expressing P protein, but not tyrosinase, do nothave detectable tyrosinase activity, showing that P proteins effect ontyrosinase activity in these cells is completely dependent on theexpression of tyrosinase.

[0131] The tyrosinase activity of cells made to express both tyrosinaseand P protein is sensitive to the action of compounds that inhibit Pprotein function. When these cells are treated with, for example,imipramine, the tyrosinase activity of these cells is markedly reduced.The effect of these compounds on tyrosinase activity is totallydependent on the presence of active P protein. Cells expressingtyrosinase but not P protein have tyrosinase activities that areunaffected by the presence of the compound at the concentrations tested.

[0132] These observations are exploited in a number of methods ofscreening for compounds that affect (e.g., decrease or increase) Pprotein function. Cells that do not otherwise have detectable tyrosinaseand/or P protein are made to express both of these proteins. The cellsare grown or incubated in medium that contains a compound to be tested.The tyrosinase activity of extracts of these cells is measured.Tyrosinase activity can be measured using any of the assays discussedabove, including the radiometric tyrosine hydroxylase assay,colorimetric DOPA oxidase assay, the DHICA converting assay, an assayfor the ability to convert [¹⁴C]DOPA into TCA precipitable material, orby any other method known in the art. If the tyrosinase activity of theextracts of cells treated with the test compound is lower than thetyrosinase activity of the extracts of similar cells grown under similarconditions but without the test compound, and if the compound does nototherwise decrease tyrosinase activity in the extracts of cellsexpressing tyrosinase but not P protein, then the compound decreases Pprotein function. Conversely, if the tyrosinase activity of the extractsof cells treated with the test compound is higher than the tyrosinaseactivity of the extracts of similar cells grown under similar conditionsbut without the test compound, and if the compound does not otherwiseincrease tyrosinase activity in the extracts of cells expressingtyrosinase but not P protein, then the compound increases P proteinfunction.

[0133] Another method of screening using non-melanogenic cells made toexpress tyrosinase and P protein exploits, in part, the discovery thatthese cells, if incubated long enough, turn black with melanindeposition. Cells expressing tyrosinase and P protein, or tyrosinase butnot P protein, are treated with a compound to be tested. The cells areincubated for a period of time sufficient to allow cells expressing bothtyrosinase and P protein, but which are not treated with the testcompound, to accumulate melanin. The melanin content of treated anduntreated cells can be assayed by visual inspection orspectrophotometric analysis of the cells, or by using other techniqueswell known in the art. If the melanin content of the cells expressingboth tyrosinase and P protein and treated with the test compound islower than the melanin content of similar cells not treated with thecompound, then the compound can decrease melanogenesis. If the melanincontent of cells expressing tyrosinase but not P protein is notsubstantially altered by the presence or absence of the compound, thenthe compound inhibits P protein function. Conversely, compounds thatcause an increase in melanin formation in these cells, relative tosimilar cells grown under similar conditions but without the compound,increase melanogenesis. If the compound also fails to increase melaninformation in non-melanogenic cells expressing a tyrosinase-encoding genebut not a P protein-encoding gene, then the compound increases P proteinfunction.

[0134] Alternatively, broken cell extract systems can be devised tostudy intracellular trafficking of tyrosinase. In a non-limitingexample, donor Golgi membranes and cytosol from wild-type melanocytescan be combined with melanosomes prepared from cells of a mouse with amutation in the tyrosinase gene that inactivates the enzyme. One couldthen observe the transfer of tyrosinase from the wild-type donor Golgimembranes to the tyrosinase-deficient melanosomes. Addition of acompound that inhibits P protein function would inhibit such transfer.

[0135] For those methods using heterologous genes, the heterologoustyrosinase and P protein-encoding genes can be derived from any suitablesource. Preferably, they are derived from an animal source. Morepreferably, they are derived from a mammalian source such as the mousecells used below in illustrating embodiments. Even more preferably, theyare derived from a primate source such as humans. Tyrosinase-encodinggenes are well known in the art (see, for example, expression of thehuman gene cDNA in Bouchard et al., 1989, J. Exp. Med. 169 (6),2029-2042, and the MEDLINE database at accession nos., for example,NM_(—)000372, M27160, and U01873), as are P protein-encoding genes (seefor example, Rinchik et al., 1993, Nature 361 (6407), 72-76, and theMEDLINE database at accession nos., for example, NM_(—)000275 and U19152for the human gene).

[0136] Expression cassettes are typically used to express heterologousgenes in the chosen cell. Each expression cassette contains regulatorysequences designed to express, for example, the tyrosinase-encoding geneand/or the P protein-encoding gene. For expression in prokaryotic cells,preferably each coding sequence found in the expression cassette isoperatively linked to at least one regulatory sequence, i.e., a promotersequence. By “operatively linked” is meant that the regulatory sequencefunctions to regulate the coding sequence (e.g., controls the timing oramount of expression of the coding sequence, determines initiation ortermination of transcription or translation, or affects messagestability). For expression in eukaryotic cells, preferably each codingsequence found in the expression cassette is “operatively linked” to atleast two regulatory sequences, i.e., a promoter and a polyA sequence.Each expression cassette is operatively linked to the polynucleotidesequence of a vector. Each vector preferably contains polynucleotidesequences that allow for its selection, replication, and maintenance intransfected cells, either as an autonomous extrachromosomal element, oras an integrated component of one or more chromosomes in the transfectedcells. Vectors containing expression cassettes that can be adapted toexpress almost any coding sequence are well known in the art andcommercially available. Non-limiting examples of such vectors areillustrated below using the pcDNA vectors available from Invitrogen (SanDiego, Calif.).

[0137] Any promoter that facilitates a sufficiently high rate ofexpression can be used in the expression cassette. The promoter can beconstitutive or inducible. See, e.g., Resendez et al., 1988, Mol. CellBiol. 8:4579-4584; and Chang et al., 1987, Proc. Natl. Acad. Sci. USA84:680-684, which describe inducible promoters. The choice of thepromoter depends on what cell type is used in the screen and the desiredlevel of expression of the heterologous genes encoding tyrosinase and/orP protein. See, e.g., Gossen et al., 1995, Science 268:1766-1769; Gossenand Bujard, 1992, Proc. Natl. Acad. Sci. USA 89:5547-5551 and U.S. Pat.Nos. 5,851,984; 5,849,997; 5,827,687; 5,811,260; 5,789,215; 5,665,578;5,512,483; 5,302,517; 4,959,313; and 4,935,352, which describe usefulpromoter sequences.

[0138] Further non-limiting examples of promoter sequences and elementsinclude the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290:304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. (USA) 78:1441-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. (USA)75:3727-3731), and the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. (USA) 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; plant expressionvectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaicvirus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871),and the promoter of the photosynthetic enzyme ribulose biphosphatecarboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter, and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion, which is active in pancreatic acinar cells (Swift et al. (1984)Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatol. 7:425-515); the insulin genecontrol region, which is active in pancreatic beta cells (Hanahan, 1985,Nature 315:115-122); the immunoglobulin gene control region, which isactive in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control region,which is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region, which isactive in liver (Pinkert et al., 1987, Genes and Devel 1:268-276); thealpha-fetoprotein gene control region, which is active in liver(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al.,1987, Science 235:53-58); the alpha 1-antitrypsin gene control region,which is active in the liver (Kelsey et al., 1987, Genes and Devel.1:161-171); the beta-globin gene control region, which is active inmyeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al.,1986, Cell 46:89-94); the myelin basic protein gene control region,which is active in oligodendrocyte cells in the brain (Readhead et al.,1987, Cell 48:703-712); the myosin light chain-2 gene control region,which is active in skeletal muscle (Sani, 1985, Nature 314:283-286); andthe gonadotropic releasing hormone gene control region, which is activein the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0139] Another regulatory element that can be used in the expressioncassette for eukaryotic cell expression is a polyA sequence (or polyAsignal), which should be capable of efficiently inducing polyadenylationof a transcript specific for the coding sequence to which the polyAsequence is operatively linked. See, e.g., U.S. Pat. Nos. 5,861,290;5,851,984; 5,840,525 and 5,627,033, which discuss polyA sequences.

[0140] In another non-limiting embodiment, the expression cassette usedaccording to the present invention may further comprise an enhancerelement, a 5′ or 3′ untranslated sequence (or region), one or moreintrons, a sequence that regulates RNA stability, or a combination ofmore than one of these elements. Any sequence that falls into any ofthese categories can be used in the vector of the present invention. SeeU.S. Pat. Nos. 5,861,290; 5,851,984; 5,840,525; 5,681,744 and 5,627,033,which discuss these regulatory elements. The term “5′ untranslatedsequence” refers to the sequence of an mRNA molecule between thetranscription initiation site and the translation initiation site. Theterm “3′ untranslated sequence” refers to the sequence of an mRNAmolecule between the translation termination site and the polyA tail.

[0141] The heterologous genes used in these assays are typicallyintroduced into the chosen cells by transfection, transduction,transformation, or any other suitable technique known in the art. Forexample, electroporation, calcium phosphate coprecipitation,microinjection, lipofection, etc., can be used. See, e.g., U.S. Pat.Nos. 5,814,618 and 5,789,215, which describe transfection methods. Thecells that take up the heterologous gene or genes, either throughintegration into their genome or by maintenance as part of anextrachromosomal element, are then preferably selected by standardtechniques. Thus, a selectable marker can be included in the vectorwhich allows a cell that has the marker, and thus cells that contain thevector and the heterologous gene or genes, to be isolated from cellsthat do not have the marker. Whether a selectable marker is necessary toprepare the cells used in these assays depends on the particular methodby which the vector is introduced into the cells. For example, if thevector is introduced into the cells via microinjection, a selectablemarker may be less useful than if electroporation is used because thetransformation frequency tends to be higher for microinjection. Forexample, the marker can enable a cell to grow under selectiveconditions, i.e. conditions under which the cell could not grow if itdid not have the marker (e.g., the neomycin resistance gene and thehypoxanthine phosphoribosyltransferase gene). A marker can also provideanother means by which to identify the cell which took up theheterologous polynucleotide molecule or vector (e.g., by preferentialstaining). See, e.g., U.S. Pat. Nos. 5,851,984 and 5,789,215, whichdescribe selectable markers.

[0142] The cells used in these assays can typically be derived from anysource. The cells used in these assays can be cells derived from amammalian animal, for example: a sheep, cow, pig, or other farm animal;a cat, dog, or other domesticated animal; a mouse, rat, or other rodent;a monkey, ape, or other primate; and most preferably a human.Alternatively, the cells used in these assays can be derived fromnon-mammalian animals such as, for example, a bird, fish, reptile,amphibian, or insect. Cells derived from animals for use in these assayscan be of any type such as, for example, fibroblasts, glial cells,keratinocytes, hepatocytes, ependymal cells, bone marrow cells,hippocampal cells, stem cells, embryonic stem cells, hematopoietic stemcells, olfactory mucosa cells, adrenal cells, leukocytes, lymphocytes,chromaffin cells, neurons, cells of the immune system, macrophages,Schwann cells, oligodendrocytes, astrocytes, germline cells, somaticcells, epithelial cells, endothelial cells, adrenal medulla cells,osteoblasts, osteoclasts, myoblasts, pancreatic cells (e.g., of theislets of Langerhans), or a mixture of more than one of the above celltypes, etc. Alternatively, the cells used in these assays can be derivedfrom a plant source such as, for example, a dicotyledon such as, e.g.,tobacco, or a monocotyledon, such as, e.g., corn. Alternatively, thecells used in these assays can be derived from a unicellular eukaryoticorganism such as, for example, a protozoan or a yeast or otherunicellular fungus. Methods of growing these cells are specific to eachcell type and within the skill of the art.

[0143] In a preferred embodiment, established cell lines from any ofthese sources can be used for these assays. Examples of suitable celllines include, but are not limited to, Chinese Hamster Ovary (CHO)cells, HeLa cells, NRK cells, A293 cells, and COS cells, which arecommercially available; e.g., from the American Type Culture Collection,Manassas, Va. The cells should have the ability to proliferate whengrown in in vitro culture. Following introduction of the heterologousgene or genes into the cells, and selection for cells that have taken upthe heterologous gene or genes, such cells, in a preferred embodiment,should be useful to establish a cell line that can be grown, stored,re-grown, etc., for extended periods of time in in vitro culture. See,e.g., U.S. Pat. No. 5,814,618, which describes cells useful for theassays of the present invention.

[0144] 1.8 High-Throughput Methods of Screening for Compounds thatAffect or Mimic P Protein Function

[0145] The methods of screening for compounds that affect or mimic Pprotein function described above can be used to test individualcompounds or small numbers or large numbers of compoundscontemporaneously. High-throughput methods of screening, as known in theart, are preferable.

[0146] For purposes of the present invention, the term “high-throughputmethod of screening” is defined as a method of screening that allows forlarge numbers of compounds to be tested concurrently. Each or all of thesteps in screening compounds that affect or mimic P protein function areamenable to high throughput methods of screening for candidatecompounds. Preferably, the high-throughput methods of screening arepartially or fully automated, reducing the amount of attention requiredto test each compound. For example, an increase in the amount oftyrosinase secreted into the medium, or total levels of tyrosinaseactivity, can be detected easily in the formats (such as, e.g., 96 wellplates) typically used in high-throughput methods of screening.High-throughput methods of screening are well known in the art and canbe performed in any of a number of formats. Laboratory automation,including robotics technology, can significantly decrease the timenecessary to screen large numbers of compounds, and is commerciallyavailable from, for example, Tecan (Research Triangle Park, N.C.),Scitec Laboratory Automation SA (Lausanne, Switzerland), Rosys (NewCastle, Del.), Rixan Associates Inc. (Dayton, Ohio), CRS Robotics(Burlington, Ontario Canada), Fanuk Robotics, and Beckman-Coulter Sagian(Indianapolis, Ind.), to name just a few companies. Upon identifyingcandidate compounds, secondary methods of screening can be performed todetermine the cellular and/or in vivo effects of the candidate compoundson P protein function.

[0147] 1.9 Secondary Methods of Screening and Additional Methods ofScreening for Compounds That Affect or Mimic P Protein Function

[0148] Each of the above methods of screening can be used by itself toidentify compounds that are likely to affect or mimic P proteinfunction. Alternatively, a plurality of methods of screening can be usedserially to confirm, or to determine more accurately, the P proteinaffecting properties of one or more compounds. For example, any of theabove methods of screening can be used as a primary method of screening,followed by a secondary method of screening. For purposes of the presentinvention, the term “primary method of screening” is defined as thefirst method of screening used to test the ability of a compound toaffect or mimic P protein function. For purposes of the presentinvention, the term “secondary method of screening” is defined as anymethod of screening that is not the primary method of screening. The useof secondary methods of screening is particularly important when theprimary method of screening is based on the identification of compoundsthat lower the activity of tyrosinase or the amount of melanin produced,or that lower the amount of tyrosinase secreted. Direct inhibitors oftyrosinase will also cause a reduction in the activity of tyrosinase andthe amount of melanin produced, or can cause a reduction in tyrosinaseactivity, but would not necessarily affect P protein function.

[0149] Any of the methods of screening described above can also be usedas a secondary method of screening. For example, one can identifycandidate compounds using as a primary screen the assay for an effect ontyrosinase activity in cells made to express tyrosinase and P protein,yet which don't affect tyrosinase activity in cells made to expresstyrosinase alone. Promising compounds from this primary screen can thenbe tested in a secondary screen in an assay for their effect on cellularlocalization of tyrosinase and/or lysosomal enzymes in melanogeniccells. Of the methods of screening described above, the ones which relyupon identification of the mislocalization of tyrosinase protein oractivity or size are preferred as secondary methods of screening.

[0150] In one embodiment, a secondary screen is employed to distinguishthe effects of test molecules that effect the melanogenic pathway ingeneral and P Protein in particular, and those that inhibit proteinsynthesis, trafficking and proteolysis. For example, in an assay foractivators of P protein function, a secondary screen can simply entailvisually examining the test melanocytes to ensure a darker color and,therefore, an increase in P protein activity, rather than a generalinhibition of protein synthesis, trafficking or proteolysis by the testmolecule and resulting decrease in tyrosinase secretion. Alternatively,the cells can be histologically examined, preferably by electronmicroscopy, optionally together with DOPA staining (as described inExample 4, infra), to determine their melanosome content. A trueactivator of P protein activity will promote the maturation ofmelanosomes from stages I-III to stages III-IV, whereas an inhibitor ofprotein synthesis, trafficking and proteolysis is unlikely to promotemelanosome maturation.

[0151] Other methods of screening can be used. For example, compoundscan first be screened for binding affinity to purified P protein.Alternatively, a compound identified by a primary method of screening asaffecting P protein function can be tested for direct binding topurified P protein in vitro, or by copurification with P protein from Pprotein-expressing cells treated with the compound. Each of thesemethods of screening can determine whether the compound binds directlyto P protein. A compound that can bind directly to P protein and whichalso affects tyrosinase activity or localization or some other aspect ofmelanogenesis is likely to directly affect P protein function.Alternatively, a compound identified by a primary method of screening asaffecting P protein function can be tested for the ability to affecttyrosinase directly. For example, the test compound can be added to asystem that contains tyrosinase but not P protein. Such a system can be,for example, an in vitro system containing purified or partiallypurified tyrosinase protein free or essentially free of P protein.Alternatively, it can be a cell that expresses tyrosinase but not Pprotein. If the effect of the test compound on tyrosinase is P proteinindependent, then the test compound does not affect P protein function.If the effect of the test compound on tyrosinase is also observed in theabsence of cellular trafficking (e.g., on purified tyrosinase protein,and not in cells), then the test compound does not mimic P proteinfunction.

[0152] While preferred primary methods of screening, especially thosethat are high-throughput methods of screening, are those with the lowestcosts (that is, can be performed as quickly, with as little humansupervision, and using as few materials as possible), secondary methodsof screening can be more time, labor, and material-intensive. This isbecause the secondary methods of screening are performed only on testcompounds that are identified by the primary method of screening asaffecting or mimicking P protein function. These compounds are expectedto be a small fraction of the total number of compounds tested in anylarge scale, high-throughput screening effort. Examples of methods ofscreening that are better suited for secondary screens than for primaryscreens include administration of a test compound to an animal (e.g.,topically, subcutaneously, or orally) or to animal skin equivalentsgrown in culture, where lightening of the skin or skin equivalentindicates that the compound inhibits P protein function. Or, forcompounds that mimic P protein function, the secondary screen caninclude administration of the test compound to a melan-p animal oranimal skin equivalent, where darkening of the skin or skin equivalentindicates that the compound mimics P protein function.

[0153] Primary and secondary methods of screening can be used in anotherway to identify compounds that affect or mimic P protein function. Oncea compound that affects or mimics P protein function is identified byusing, for example, a primary method of screening, chemical analogs ofthe compound can be selected or created. For purposes of the presentinvention, the term “chemical analog” is defined as a compound that ischemically related to another chemical compound. The relationship ispreferably structural as known in the art such as where, for example,the two compounds differ only in the location of a substituent, such as,e.g., a hydroxyl or alkyl group, or are chemical homologs of each other.Alternatively, the relationship might be functional such as where, forexample, both compounds affect the same mechanism, such as, e.g., whereboth compounds are kinase inhibitors. Methods for designing or selectingchemical analogs are described below in Section 2.3. These chemicalanalogs can then be tested for the ability to affect or mimic P proteinfunction using, for example, any method described above. The secondarymethod of screening can be the same as the primary method of screening,or it can be a different method of screening. Chemical analogs aresought which have a stronger effect on P protein function than theoriginal test compound. This procedure can be repeated serially toidentify or create compounds of increasing efficacy.

[0154] 1.10 Other Compounds Identified by the Methods of Screening

[0155] The methods of screening of the invention as described herein,supra, are also useful and easily adaptable by those skilled in the artfor the identification of other compounds that inhibit or inducemelanogenesis. By way of a non-limiting example, the methods ofscreening of the invention may be used to identify compounds that effectan alteration in late endosomal/lysosomal trafficking, and therefore,reduce or inhibit melanogenesis and/or pigmentation. Such compounds arealso useful as skin lightening agents.

[0156] In a certain embodiment, the methods of screening may be used toidentify compounds that effect an alteration in late endosomal/lysosomalcholesterol trafficking and thereby reduce or inhibit melanogenesisand/or pigmentation. Such compounds are also useful as skin lighteningagents. Intracellular cholesterol originates from two sources: (1)endocytosis of plasma lipoproteins, which contain free and esterifiedcholesterol and triglycerides; and (2) endogenous synthesis of freecholesterol in the endoplasmic reticulum (ER). Both endocytosedcholesterol and endogenously synthesized cholesterol are traffickedwithin the cell and ultimately are distributed to various membranes,stored as cholesterol ester in lipid droplets, or exported in plasmalipoproteins. In general, the trafficking of cholesterol involvesendocytosis of plasma lipoproteins that are delivered to lateendosomes/lysosomes for hydrolysis of esterified cholesterol andmovement of subsequent free cholesterol from the late endosome/lysosometo the ER or plasma membrane (PM). From the ER, cholesterol may betrafficked to the trans-Golgi network (TGN), and from there to theplasma membrane. Cholesterol can also move from the PM back to the ER(Liscum et al. (1999) Biochim. Biophys. Act 1438:19-37). Although exactmechanisms involved in late endosomal/lysosomal cholesterol traffickingare not well understood, at least one protein is specifically known tobe involved. Defective Niemann Pick type C1(NPC1) protein has been slowto demonstrate the movement of cholesterol to the PM and to the ER,trapping free cholesterol in a lysosomal or a cholesterol-sortingcompartment (Cruz et al. (2000) J. BioL Chem. 275:4013-21).

[0157] The screening methods of the invention may by utilized toidentify compounds that effect an alteration in late endosomal/lysosomaltrafficking, and, in some instances, effect an alteration in lateendosomal/lysosomal cholesterol trafficking.

[0158] 2. Compounds for Inhibiting or Inducing Melanogenesis

[0159] 2.1 Compounds for Inhibiting, Increasing or Mimicking P ProteinFunction

[0160] Compounds that can be screened in accordance with the presentinvention include but are not limited to small organic molecules thatare able to gain entry into a cell and affect P protein activity. Anumber of compound libraries are commercially available from companiessuch as Pharmacopeia (Princeton, N.J.), Arqule (Medford, Mass.), Enzymed(Iowa City, Iowa), Sigma-Aldrich (St. Louis, Mo.), Maybridge(Trevillett, United Kingdom), Trega (San Diego, Calif.) and PanLabs(Bothell, Wash.), to name just a few sources. One also can screenlibraries of known compounds, including natural products or syntheticchemicals, and biologically active materials, including proteins, forcompounds that affect or mimic P protein function.

[0161] One class of preferred compounds for use in the methods of thepresent invention comprises chemical analogs of imipramine. As describedabove, imipramine inhibits P protein function. Imipramine is a tricyclictertiary amine used in the treatment of depression. See Gilman, A.G. etal., eds, 1990, Goodman and Gilman's The Pharmacological Basis ofTherapeutics, Eighth Edition, 405-14, Pergamon Press, New York. Othertricyclic tertiary amines used in the treatment of depression such as,for example, amitriptyline, trimipramine, or doxepin (Sigma, St. Louis,Mo.) (see id.) can be test compounds in screens for compounds thataffect P protein function. Secondary amines used in the treatment ofdepression such as, for example, desipramine, nortriptyline,protriptlyine, amoxapine, or maprotiline (Sigma, St. Louis, Mo.) (seeid.) also are preferred compounds for the screens of the presentinvention. These chemical analogs of imipramine all share structural andfunctional characteristics with imipramine. Other chemical analogs ofimipramine that are preferred compounds for use in the methods of thepresent invention include chemicals with functional and/or structuralsimilarities to imipramine. For example, the atypical antidepressantssuch as, for example, trazodone and fluoxetine (Sigma, St. Louis, Mo.),lack structural similarity with imipramine (see id.), but share thefunctional property with imipramine of being useful antidepressants, andso are preferred compounds for the screens of the present invention.Tricyclic compounds, tertiary amines, and secondary amines withoutantidepressant effects also are preferred compounds of the presentinvention.

[0162] Another class of compounds that can be used to inhibit thefunction of P protein are P protein-encoding gene antisense nucleicacids. A P protein-encoding gene antisense nucleic acid as used hereinrefers to an oligonucleotide or polynucleotide molecule having a nucleicacid sequence capable of hybridizing to a portion of a Pprotein-encoding RNA (preferably mRNA) by virtue of some degree ofsequence complementarity. The antisense nucleic acid should becomplementary to either a coding and/or noncoding region of a P proteinmRNA such that it inhibits P protein function by reducing the amount ofP protein synthesized.

[0163] The antisense nucleic acids of the present invention can beoligonucleotides that are double-stranded or single-stranded, RNA orDNA, or a modification or analog thereof, which can be directlyadministered to a cell, or to the skin of an animal, or which can beproduced intracellularly by transcription of heterologous, introducedsequences.

[0164] In one embodiment, the present invention is directed to methodsfor inhibiting the expression of a P protein-encoding nucleic acidsequence in a prokaryotic or eukaryotic cell comprising providing thecell with an effective amount of a composition comprising a Pprotein-encoding gene antisense nucleic acid of the present invention.

[0165] The P protein-encoding gene antisense nucleic acids of thepresent invention are at least about six nucleotides in length and aremore preferably oligonucleotides ranging from about 6 to about 50oligonucleotides. In specific aspects, the oligonucleotide is at leastabout 10 nucleotides, at least about 15 nucleotides, at least about 100nucleotides, or at least about 200 nucleotides in length. Theoligonucleotides can be DNA or RNA, or chimeric mixtures or derivatives,and modified versions thereof, which can either be single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone level. The oligonucleotide mayinclude other appending groups such as peptides, or agents facilitatingtransport across the cell membrane (see, e.g., Letsinger et al., 1989,Proc. Natl. Acad. Sci. (USA) 86:6553-6556; Lemaitre et al., 1987, Proc.Natl. Acad. Sci. (USA) 84:648-652; PCT Publication No. WO 88/09810,published Dec. 15, 1988), hybridization-triggered cleavage agents (see,e.g., Krol et al., 1988, BioTechniques 6:958-976), or intercalatingagents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). In a preferredaspect of the present invention, a P protein-encoding gene antisenseoligonucleotide is a single-stranded DNA molecule.

[0166] The oligonucleotide may be modified at any position on itsstructure with substituents generally known in the art. The Pprotein-encoding gene antisense oligonucleotide may comprise at leastone modified base moiety which is selected from a group including, butnot limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[0167] In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety selected from a group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose. In yet anotherembodiment, the oligonucleotide comprises at least one modifiedphosphate backbone component selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof. In yet anotherembodiment, the oligonucleotide is an alpha-anomeric oligonucleotide. Analpha-anomeric oligonucleotide forms specific double-stranded hybridswith complementary RNA in which, contrary to the usual beta-units, thestrands run parallel to each other (Gautier et al., 1987, Nucl. AcidsRes. 15:6625-6641).

[0168] The oligonucleotide may be conjugated to another molecule suchas, e.g., a peptide, hybridization triggered cross-linking agent,transport agent, hybridization-triggered cleavage agent, etc.

[0169] Oligonucleotides of the present invention may be synthesized bystandard methods known in the art including, e.g. by use of an automatedDNA synthesizer (such as are commercially available from Biosearch,Applied Biosystems, etc.). As examples, phosphorothioateoligonucleotides may be synthesized by the method of Stein et al., 1988,Nucl. Acids Res. 16:3209, and methylphosphonate oligonucleotides can beprepared by use of controlled pore glass polymer supports using themethod of Sarin et al., 1988, Proc. Natl. Acad. Sci. (USA) 85:7448-7451,etc.

[0170] In a specific embodiment, the P protein antisense oligonucleotidecomprises catalytic RNA, or a ribozyme (see, e.g., PCT InternationalPublication WO 90/11364, published Oct. 4, 1990; Sarver et al., 1990,Science 247:1222-1225). In another embodiment, the oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0171] In an alternative embodiment, the P protein-encoding geneantisense nucleic acid of the invention is produced intracellularly bytranscription from an heterologous sequence. For example, a vector canbe introduced in vivo such that it is taken up by a cell, within whichcell the vector or a portion thereof is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the P protein-encoding gene antisensenucleic acid. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA. Such vectors can be constructed by standard recombinantDNA technology methods known in the art. Vectors can be plasmids, viralvectors, or others known in the art as useful for replication andexpression in mammalian cells. Expression of the sequence encoding the Pprotein-encoding gene antisense RNA can be regulated by any promoterknown in the art to act in such cells. Such promoters can be inducibleor constitutive, and can include but are not limited to those listedabove.

[0172] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a Pprotein-encoding gene, preferably a human P protein-encoding gene.However, absolute complementarity, although preferred, is not required,as long as the antisense nucleic acid has sufficient complementarity tobe able to hybridize with the RNA, forming a stable duplex. In the caseof double-stranded P protein-encoding gene antisense nucleic acids, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with a P protein-encoding gene RNA it may contain and stillform a stable duplex (or triplex, as the case may be). One skilled inthe art can determine the mismatch tolerance by use of standardprocedures to, e.g., determine the melting point of the hybridizedcomplex.

[0173] Compounds effective at increasing or mimicking P protein functioninclude bafilomycin A1 and concanamycin A. Bafilomycins andconcanamycins are unusual macrolide antibiotics which have been isolatedfrom Streptomyces (Drose, S. et al. (1997) J. Exp. Biol 200:1-8). Thesecompounds have been shown to inhibit the activity of vacuolar ATPases(V-ATPases) at nanomolar concentrations and ATPases with phosphorylatedstates (P-ATPases) at micromolar concentrations (Bowman, EJ. et al.(1988) Proc. Natl. Acad. Sci. (USA) 85:7972-6). Furthermore, bafilomycinA1 and concanamycin A and B have been shown to inhibit p-glycoproteinATPase activity at micromolar concentrations (Sharom, F.J. et al (1995)Biochem. J. 308:381-90). At nanomolar concentrations, bafilomycin Al andconcanamycin A activate melanogenesis in certain amelanotic melanomacells that nonetheless express tyrosinase (Ancans, J. et al. (2000) FEBSLett. 478:57-60).

[0174] The present invention provides for these compounds and analogsthereof for the activation of melanogenesis in melan-p-melanocytes.Bafilomycin A1 and concanamycin A may be isolated from Streptomyces aspreviously described or may be obtained from Sigma (St. Louis, Mo.).

[0175] 2.2 Compounds that Inhibit Late Endosomal/lysosomal Trafficking

[0176] Compounds that can be screened in accordance with the presentinvention include, but are not limited to, small organic molecules thatare able to gain entry into a cell and alter or inhibit lateendosomal/lysosomal trafficking. A number of compound libraries arecommercially available from companies such as Pharmacopeia (Princeton,N.J.), Arqule (Medford, Mass.), Enzymed (Iowa City, Iowa), Sigma-Aldrich(St. Louis, Mo.), Maybridge (Trevillett, United Kingdom), Trega (SanDiego, Calif.) and PanLabs (Bothell, Wash.), to name just a few sources.Libraries of known compounds, including natural products or syntheticchemicals, and biologically active materials, including proteins, forcompounds that alter or inhibit late endosomal/lysosomal trafficking mayalso be screened.

[0177] An alteration in late endosomal/lysosomal trafficking may beeffected by contacting the melanocyte with a compound such asprogesterone, a hydrophobic amine, sphingosine, an antagonist of a lateendosomal/lysosomal trafficking protein, or a compound of the formula

[0178] wherein X is O or S. In a preferred embodiment, X is O.

[0179] R₁ is —C(O)(C₁-C₆)alkyl or —(CH₂)_(n)—O—(C₁-C₆)alkyl, or—(CH₂)_(n)—NR₇R₈ where n is 0-3, where each of R₇ and R₈ areindependently selected from H and (C₁-C₆)alkyl. Preferably, each of R₇and R₈ are independently selected from —C(O)(C₁-C₃)alkyl,—CH₂—O—(C₁-C₃)alkyl, or —(CH₂)₂—N(C₁-C₃ alkyl)₂. More preferably, eachof R₇ and R₈ is independently selected from —C(O)CH₃,—CH₂—O—CH₃, or—(CH₂)₂—N(CH₃)₂.

[0180] R₂ is H or (C₁-C₆)alkyl. In some embodiments, R₂ is (C₁-C₃)alkyl.In preferred embodiments, R₂ is —CH₃.

[0181] R₃ is H or (C₁-C₆)alkyl. In preferred embodiments, R₃ is(C₁-C₃)alkyl. In more preferred embodiments, R₃ is —CH₃.

[0182] R₄ is —C(O)(C₁-C₆)alkyl. Preferably, R₄ is —C(O)(C₁-C₃)alkyl. Inmore preferred embodiments, R₄ is —C(O)CH₃ or —C(O)CH₂CH₃.

[0183] R₅ is H or —(C₁-C₆)alkyl. Preferably, R₅ is H or —CH₃. In anotherembodiment, R₄ and R₅ taken together are ═O.

[0184] R₆ is H or —(C₁-C₆)alkyl or —(CH₂)_(n)—NR₉R₁₀, where each of R₉and R₁₀ are independently selected from H and (C₁-C₆)alkyl. Preferably,R₆ is H or —CH₃, or —CH₂CH₃ or —CH₂NH₂. In another embodiment, R₅ and R₆are combined with the carbon atoms to which they are attached to form aC₅-C₈ carbocyclic ring, and preferably a C₆ carbocyclic ring, which canbe substituted by one to three of halogen, OH, —(C₁-C₆)alkyl,—(C₁-C₆)alkoxy, amino, ═O, (C₁-C₆)alkylamino, di-((C₁-C₆)alkyl)amino,trifluoromethyl, or —OCF₃, which substituents can be substitutedanywhere on the carbocyclic ring where it is possible to make suchsubstitutions.

[0185] The term “alkyl”, as used herein, unless otherwise indicated,includes saturated monovalent hydrocarbon radicals having straight,branched or cyclic moieties or combinations thereof. Any substituents orfunctional groups on the alkyl group, as indicated herein, can besubstituted anywhere on the alkyl group.

[0186] The term “halo”, as used herein, refers to halogen and, unlessotherwise indicated, includes chloro, fluoro, bromo and iodo.

[0187] Compounds of formula I may contain chiral centers and, therefore,may exist in different enantiomeric and diastereomeric forms. Thisinvention relates to all optical isomers, stereoisomers and tautomers ofthe compounds of formula I, and mixtures thereof, and to allpharmaceutical compositions and methods of treatment defined above thatcontain or employ them, respectively.

[0188] Formula I, as defined above, also includes compounds identical tothose depicted but for the fact that one or more hydrogen, carbon orother atoms are replaced by isotopes thereof. Such compounds may beuseful as research and diagnostic tools in metabolism pharmacokineticstudies and in binding assays.

[0189] The present invention also relates to the pharmaceuticallyacceptable acid addition and base salts of any of the aforementionedcompounds of formula I. The acids which are used to prepare thepharmaceutically acceptable acid addition salts of the aforementionedbase compounds of this invention are those which form non-toxic acidaddition salts, ie., salts containing pharmacologically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate,citrate, acid citrate, tartrate, bitartrate, succinate, maleate,fumarate, gluconate, saccharate, benzoate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,1,1-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

[0190] The compounds useful according to the invention that are basic innature are capable of forming a wide variety of different salts withvarious inorganic and organic acids. Although such salts must bepharmaceutically acceptable for administration to animals, it is oftendesirable in practice to initially isolate a compound of formula I fromthe reaction mixture as a pharmaceutically unacceptable salt and thensimply convert the latter back to the free base compound by treatmentwith an alkaline reagent and subsequently convert the latter free baseto a pharmaceutically acceptable acid addition salt. The acid additionsalts of the active base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained.

[0191] Those compounds useful according to the invention that are acidicin nature are capable of forming base salts with variouspharmaceutically acceptable cations. Examples of such salts include thealkali metal and alkaline earth metal salts and, particularly, thesodium and potassium salts. These salts can be prepared by conventionaltechniques. The chemical bases that are used as reagents to prepare thepharmaceutically acceptable base salts of this invention are those thatform non-toxic base salts with the acidic compounds of formula I. Suchnon-toxic base salts include those derived from such pharmaceuticallyacceptable cations as sodium, potassium, calcium and magnesium, etc.These salts can easily be prepared by treating the corresponding acidiccompounds with an aqueous solution containing the desiredpharmaceutically acceptable cations, and then evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively,they can also be prepared by mixing lower alkanolic solutions of theacidic compounds and the desired alkali metal alkoxide together, andthen evaporating the resulting solution to dryness, as described above.In either case, stoichiometric quantities of reagents are preferablyemployed in order to ensure completeness of reaction and maximum yieldsof the desired final products.

[0192] Compounds of generic formula (I) are disclosed and may besynthesized following procedures detailed in WO 00/58549 and U.S. Pat.Nos. 5,232,917, 3,210,386, 3,389,051, and 2,794,815.

[0193] The compounds useful according to the invention, and theirpharmaceutically acceptable salts, are useful in the treatment ofdisorders of human pigmentation, including solar and simple lentigines(including age/liver spots), melasma/chloasma and postinflammatoryhyper-pigmentation. Such compounds reduce skin melanin levels byinhibiting the production of melanin, whether the latter is producedconstitutively or in response to UV irradiation (such as sun exposure).Thus, some of the active compounds used in this invention can be used toreduce skin melanin content in non-pathological states so as to induce alighter skin tone, as desired by the user, or to prevent melaninaccumulation in skin that has been exposed to UV irradiation. They canalso be used in combination with skin peeling agents (including glycolicacid or trichloroacetic acid face peels) to lighten skin tone andprevent repigmentation. Other compounds useful according to theinvention, and their pharmaceutically acceptable salts, are useful inthe treatment of skin conditions where insufficient skin pigmentation isproduced, or where the subject, for cosmetic purposes, simply wishes todevelop a “sunless tan”.

[0194] The appropriate dose regimen, the amount of each doseadministered, and specific intervals between doses of the activecompound will depend upon the particular active compound employed, thecondition of the patient being treated, and the nature and severity ofthe disorder or condition being treated. Preferably, the active compoundis administered in an amount and at an interval that results in thedesired treatment of or improvement in the disorder or condition beingtreated.

[0195] For skin lightening, an active compound used in the presentinvention can also be used in combination with sun screens (UVA or UVBblockers) to prevent repigmentation, to protect against sun orUV-induced skin darkening or to enhance their ability to reduce skinmelanin and their skin bleaching action. For skin lightening, an activecompound used in the present invention can also be used in combinationwith retinoic acid or its derivatives or any compounds that interactwith retinoic acid receptors and accelerate or enhance the invention'sability to reduce skin melanin and skin bleaching action, or enhance theinvention's ability to prevent the accumulation of skin melanin. Forskin lightening, an active compound used in the present invention canalso be used in combination with 4-hydroxyanisole. For skin lightening,the active compounds used in this invention can also be used incombination with ascorbic acid, its derivatives and ascorbic-acid basedproducts (such as magnesium ascorbate) or other products with ananti-oxidant mechanism (such as resveratrol) which accelerate or enhancetheir ability to reduce skin melanin and their skin bleaching action.

[0196] Non-limiting examples of compounds that cause an alteration inlate endosomal/lysosomal trafficking of cholesterol include U18666A(formula VlI) and its derivatives (e.g., formulae II-VI), either aloneor in combination. Useful examples of U18666A derivatives includeCP-598755-01 (formula III), CP-602367 (formula IV), CP-352369 (formulaII), UK-204039 (formula V), UK-204041 (formula (VI), and UK-204042(formula VII). Generic formula I presented herein is derived fromU18666A and its derivatives presented herein (also see FIG. 16).

[0197] Progesterone is another compound useful in the methods andcompositions of the invention to decrease melanin production or toreduce skin pigmentation. Progesterone may be obtained from a variety ofsources, e.g., Sigma (St. Louis, Mo.).

[0198] Alternatively, hydrophobic amines may be utilized in the methodsand compositions of the invention. By the term “hydrophobic amine” ismeant a compound having structure comprising a hydrophobic ringstructure with a hydrophilic side chain containing a cationic aminefunctional group. Preferred hydrophobic amines include phenothiazinesand tricyclic antidepressants. Particularly preferred phenothiazinesinclude trifluoperazine, chlorpromazine, prochlorperazine,triflupromazine, promazine, thioridazine, mesoridaine, piperacetazine,perphenazine, fluphenazine, acetophenazine, and thiethylperazine.(Sigma, St. Louis, Mo.). Tricyclic antidepressants are other preferredhydrophobic amines that can be utilized in the methods and compositionsof the invention. Particularly preferred tricyclic antidepressantsinclude imipramine, nortriptyline, protriptyline, trimipramine, anddoxepin (Sigma, St. Louis, Mo.).

[0199] Another compound useful in the methods and compositions of theinvention is sphingosine, which is commercially available from, e.g.,Sigma (St. Louis, Mo.).

[0200] Antagonists of a late endosomal/lysosomal trafficking protein arealso useful in the methods and compositions of the invention to decreasemelanin production or to reduce skin pigmentation. By the phrase“antagonist of a late endosomal/lysosomal trafficking protein” is meantan agent that interferes with or reduces the activity of a proteininvolved directly or indirectly with late endosomal/lysosomalcholesterol trafficking and that results in an alteration in thistrafficking. By way of a non-limiting example, the agent that alterslate endosomal/lysosomal trafficking may be a small organic molecule, ora protein, or a polysaccharide, etc.

[0201] By way of a non-limiting example, an antagonist of lateendsomsomal/lysosomal trafficking may be a protein, for example, anantibody, that binds exclusively to a trafficking protein, proteolipid,proteoglycan, etc. (see Kobayashi et al. (1999) Nature Cell Biol.1:113-116, which discloses an antibody that specifically bindsphospholipid lysobiphosphatidic acid as an antagonist to cholesteroltrafficking).

[0202] The production of antibodies against specific antigenicdeterminants is well known in the art and is specifically described inCurrent Protocols in Immunology, Coligan et al. eds., (2000) John Wiley& Sons, New York, N.Y., and in Harlow & Lane, Antibodies: A LaboratoryManual (1988) Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0203] The invention also provides compounds useful to decrease melaninproduction or to reduce skin pigmentation of the formula

[0204] wherein X and R₁-R₆ are as described supra. Specific compounds offormula (I) include

[0205] and any pharmaceutically acceptable salts or solvates thereof.

[0206] 2.3 Rational Drug Design

[0207] Compounds identified by the methods of the invention or compoundsdisclosed herein may serve as the basis for molecular modelingtechniques for the design of chemical analogs that are more effective.For example, chemical analogs of imipramine, or any of the otherpreferred compounds listed above, can be created using these or othermodeling techniques. Examples of molecular modeling systems are theCHARM (Polygen Corporation, Waltham, Mass.) and QUANTA (MolecularSimulations Inc., San Diego, Calif.) programs. CHARM performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

[0208] For example, once a compound that affects or mimics P proteinfunction and/or alters or inhibits late endosomal trafficking isidentified, the compound can be used to generate a hypothesis. Such ahypothesis can be generated from any one of the preferred compounds ofthe present invention using, e.g., the program, Catalyst (MolecularSimulations Inc., San Diego, Calif.). Furthermore, Catalyst can use thehypothesis to search proprietary databases such as, for example, theCambridge small molecule database (Cambridge, England), as well as otherdatabases or compound libraries, e.g., those cited above, to identifyadditional examples of the compounds of the present invention.

[0209] Compounds of the present invention can further be used to designmore effective analogs using modeling packages such as Ludi, Insight II,C²-Minimizer and Affinity (Molecular Simulations Inc., San Diego,Calif.). A particularly preferred modeling package is MacroModel(Columbia University, New York, N.Y.).

[0210] The compounds of the present invention can further be used as thebasis for developing a rational combinatorial library. Such a librarycan also be screened to identify more effective compounds. While thenature of the combinatorial library is dependent on various factors suchas the particular compound chosen from the preferred compounds of thepresent invention to form the basis of the library, as well as thedesire to synthesize the library using a resin, it will be recognizedthat the compounds of the present invention provide requisite datasuitable for combinatorial design programs such as C²-QSAR (MolecularSimulations Inc., San Diego, Calif.).

[0211] 3. Methods of Inhibiting or Inducing Melanogenesis

[0212] 3.1 Methods of Inhibiting Melanogenesis by Increasing orMimicking P Protein Function

[0213] Compounds that affect or mimic the function of P protein can beused to treat animals or, preferably, humans that have diseases,conditions, or disorders caused by the production or overproduction ofmelanin. Such diseases, conditions, or disorders include those that canbe characterized by discolorations of the skin or hair such as, forexample, hyperpigmentation caused by inflammation or from diseases suchas melasma, or brown spots such as “café au lait” macules.Alternatively, a subject may wish to lighten the color of his or herhair or skin. Compounds that increase the function of P protein or thatmimic the function of P protein can be used to treat animals or,preferably, humans that have diseases, conditions, or disorders causedby the underproduction of melanin such as, for example,post-inflammatory hypopigmentation, pityriasis alba, and certain formsof albinism such as, for example, OCA II albinism. Additionally, suchcompounds can be used to darken the color of one's hair or skin. For thepurposes of this application, the terms “treatment”, “therapeutic use”,and “medicinal use” shall refer to any and all uses of the compositionsof the invention which remedy a disease state or one or more symptoms,or otherwise prevent, hinder, retard, or reverse the progression ofdisease or one or more other undesirable symptoms in any way whatsoever.

[0214] 3.2 Methods of Inhibiting Melanogenesis by Altering or InhibitingLate Endosomal/lysosomal Trafficking

[0215] The invention further provides methods and pharmaceuticalcompositions for inhibiting skin pigmentation comprising the use ofagents that modify late endosomal/lysosomal trafficking. These methodsand pharmaceutical compositions are useful for decreasing and/orinhibiting melanin production and, therefore, for reducing skinpigmentation. These agents may be used singly, in combination with oneanother, or in combination with other drugs that inhibit pigmentation.By way of a non-limiting example, other drugs that inhibit pigmentationinclude agents such as tyrosinase inhibitors. Preferably, the methodsand compositions of the invention are for application to a vertebrate,more particularly to a mammal, and most preferably to a human.

[0216] Several pharmacological agents are known to inhibit or alter lateendosomal/lysosomal trafficking. Hydrophobic amines, such as U18666A,progesterone, and sphingosine inhibit cholesterol movement from the PMto the ER, from the late endosome/lysosome to the ER, and from the lateendosome/lysosome to the PM (Liscum et al. supra). These agents serve asa pharmacological model for the defective NPC1 protein (Lange et al(1998) J. Biol. Chem. 273:18915-22; Roff et al. (1991) Dev. Neurosci.13:315-19). Furthermore, trafficking of the NPC1 protein itself isaltered by U18666A, shifting the protein from late endosomes to thetrans-Golgi network and lysosomes (Higgins et al. (1999) Mol. Gen.Metab. 68:1-13). U18666A has also been shown to alter the trafficking ofCD63/lamp3, a protein normally associated with the internal membranes ofmultivesicular-multilamellar late endosomes and IGF2/MPR normallyassociated with the trans-Golgi network (Kobayashi et al. (2000) Mol.Biol. Cell. 11:1829-43; Kobayashi et al. (1999) Nat Cell Biol. 1:113-118). The exact mechanism by which these agents alter lateendosomal/lysosomal trafficking is not yet understood.

[0217] The invention takes advantage of the discovery that theinhibition of late endosomal/lysosomal trafficking results in a decreasein melanin production in melanocytes. Accordingly, in one aspect, theinvention provides a method of decreasing melanin production in amelanocyte, comprising contacting the melanocyte with a pharmaceuticallyeffective amount of a compound that effects an alteration in lateendosomal/lysosomal trafficking in the melanocyte. The resultingalteration in late endosomal/lysosomal trafficking brings about adecrease in melanin production in the melanocyte.

[0218] The term “about” is used herein to mean approximately, roughly,around, or in the region of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent.

[0219] By the phrase “decrease in melanin production” is meant adetectable lowering of the amount of melanin synthesized de novo by amelanocyte exposed to a compound that alters late endosomal/lysosomaltrafficking, as compared with the amount of melanin synthesized de novoby a control, untreated melanocyte. The term “lowering” preferablyrefers to about 10% to about 100% decrease in the amount of melaninsynthesized de novo. More preferably, the term “lowering” refers toabout a 25% to about a 100% decrease in the amount of melaninsynthesized de novo. Most preferably, the term “lowering” refers toabout a 50% to about a 100% decrease in the amount of melaninsynthesized de novo.

[0220] The term “late endosomal/lysosomal trafficking” is used herein torefer to the movement of proteins, lipids, or other compounds betweendifferent cellular compartments. These locations include the movement ofsuch compounds from the late endosome to the lysosome, from the lysosometo the late endosome, from the late endosome or lysosome to the transGolgi network, and from the trans Golgi network to the late endosome orlsysome.

[0221] An alteration in late endosomal/lysosomal trafficking may beeffected by contacting the melanocyte with a compound such asprogesterone, a hydrophobic amine, sphingosine, an antagonist of lateendosomal/lysosomal trafficking, or any of the compounds of the formulae(I)-(VIII) as defined supra.

[0222] Compounds of generic formula (I) are disclosed and may besynthesized following procedures detailed in WO00/58549 and U.S. Pat.Nos. 5,232,917, 3,210,386, 3,389,051, and 2,794,815.

[0223] As one skilled in the art would know in view of this disclosure,the compounds used in the methods of the present invention may be usedalone or in combination with each other. Moreover, the methods of theinvention also include the additional use of other compounds known inthe art to affect melanin synthesis such as tyrosinase inhibitors. Suchinhibitors are known to those skilled in the art and include variousderivatives of resorcinol, hydroquinone, kojic acid, melamine, andvarious types of plant extracts, among others.

[0224] Thus, the invention relates both to methods of modulating thepigmentation of skin in which the active compound used according to theinvention, or a pharmaceutically acceptable salt thereof, and one ormore of the other active ingredients referred to above are administeredtogether, as part of the same pharmaceutical composition, as well asmethods in which they are administered separately as part of anappropriate dose regimen designed to obtain the benefits of thecombination therapy. The appropriate dose regimen, the amount of eachdose administered, and specific intervals between doses of each activeagent will depend upon the specific combination of active agentsemployed, the condition of the patient being treated, and the nature andseverity of the disorder or condition being treated. Such additionalactive ingredients will generally be administered in amounts less thanor equal to those for which they are effective as single topicaltherapeutic agents. The FDA approved dosages for such active agents thathave received FDA approval for administration to humans are publiclyavailable.

[0225] For example, any of the compounds used according to askin-lightening method of the invention may be used in combination witha tyrosinase inhibitor or other skin-whitening agent as currently knownin the art or to be developed in the future, including any one or moreof those agents described in the following patent publications: U.S.Pat. No. 4,278,656 to Nagai et al, issued Jul. 14, 1981; U.S. Pat. No.4,369,174 to Nagai et al., issued Jan. 18, 1983; U.S. Pat. No. 4,959,393to Torihara et al., issued Sep. 25, 1990; U.S. Pat. No. 5,580,549 toFukuda et al., issued Dec. 3, 1996; U.S. Pat. No. 6,123,959 to Jones etal., issued Sep. 26, 2000; U.S. Pat. No. 6,132,740 to Hu, issued Oct.17, 2000; U.S. Pat. No. 6,159,482 to Tuloup et al., issued Dec. 12,2000; WO 99/32077 by L'Oreal, published Jul. 1, 1999; WO 99/64025 byFytokem Prod. Inc., published Dec. 16, 1999; WO 00/56702 by Pfizer Inc.,published Sep. 28, 2000; WO 00/76473 by Shiseido Co. Ltd., publishedDec. 12, 2000; EP 997140 by L'Oreal SA, published May 3, 2000; JP5221846 by Kunimasa Tomoji, published Aug. 31, 1993; JP 7242687 byShiseido Co. Ltd., published Sep. 19, 1995; JP 7324023 by Itogawa H,published Dec. 12, 1995; JP 8012552 by Shiseido Co. Ltd., published Jan.16, 1996; JP 8012554 by Shiseido Co. Ltd., published Jan. 16, 1996; JP8012557 by Shiseido Co. Ltd., published Jan. 16, 1996; JP 8012560 byShiseido Co. Ltd., published Jan. 16, 1996; JP 8012561 by Shiseido Co.Ltd., published Jan. 16, 1996; JP 8134090 by Fujisawa, published May28,1996; JP 8168378 by Kirinjo KK, published Jul. 2, 1996; JP 8277225 byKansai Koso KK, published Oct. 22, 1996; JP 9002967 by Sanki Shoji KK,published Jan. 7, 1997; JP 9295927 by Yagi Akira, published Nov. 18,1997; JP 10072330 by Kansai Kouso, published Mar. 17, 1998; JP 10081626by Kamiyama KK, published Mar. 31, 1998; JP 10101543 by Kansai Kouso KK,published Apr. 21, 1998; JP 11071231 by Maruzen Pharm., published Mar.16, 1999; JP 11079934 by Kyodo Nyugyo, published Mar. 23, 1999; JP11246347 by Shiseido Co. Ltd., published Sep. 14, 1999; JP 11246344 byShiseido Co. Ltd., published Sep. 14, 1999; JP 2000-080023 by KaneboLtd., published Mar. 21, 2000; JP 2000-095663 by Kose KK, published Apr.4, 2000; JP 2000-159681 by Hai Tai Confectionary Co. Ltd., publishedJun. 13, 2000; JP 2000-247907 by Kanebo Ltd., published Sep. 12, 2000;JP-9002967 by Sanki Shoji KK, published Jan. 7, 1997; JP-7206753 byNikken Food KK, published Aug. 8, 1995; JP-5320025 by Kunimasa T,published Dec. 3, 1993; and JP-59157009 by Yakurigaku Chuou KE,published Sep. 6, 1984; among others; which patent publications areincorporated herein by reference.

[0226] Any of the compounds used according to a skin-darkening method ofthe invention may be used in combination with “sunless tanning” agentsas currently known in the art or to be developed in the future,including any one or more of those agents described in the followingpatent publications: U.S. Pat. No. 5,591,423 to Fuller, issued Jan. 7,1997; U.S. Pat. No. 5,628,987 to Fuller, issued May 13, 1997; EP 993826by L'Oreal, published Apr. 19, 2000; and WO 99/56740 by Galderma Res. &Dev., published Nov. 11, 1999; among others; which patent publicationsare incorporated herein by reference.

[0227] Non-limiting examples of compounds that cause an alteration inlate endosomal/lysosomal trafficking include U18666A and its derivatives(e.g.,formulae II-VII), either alone or in combination. Particularderivatives have been described above. Progesterone is another compounduseful in the methods and compositions of the invention to decreasemelanin production or to reduce skin pigmentation.

[0228] Alternatively, hydrophobic amines may be utilized in the methodsand compositions of the invention. By the term “hydrophobic amine” ismeant a compound having structure comprising a hydrophobic ringstructure with a hydrophilic side chain containing a cationic aminefunctional group. Preferred hydrophobic amines include phenothiazinesand tricyclic antidepressants. Particularly preferred phenothiazinesinclude trifluoperazine, chlorpromazine, prochlorperazine,triflupromazine, promazine, thioridazine, mesoridaine, piperacetazine,perphenazine, fluphenazine, acetophenazine, and thiethylperazine, asdescribed above.

[0229] Tricyclic antidepressants are other preferred hydrophobic aminesto be utilized in the methods and compositions of the invention.Particularly preferred tricyclic antidepressants are imipramine,nortriptyline, protriptyline, trimipramine, and doxepin, as describedabove. Another compound useful in the methods and compositions of theinvention is sphingosine.

[0230] In another aspect, the invention provides a method of reducingskin pigmentation. In this method the skin is contacted with apharmaceutically effective amount of a compound that effects analteration in late endosomal/lysosomal trafficking, wherein analteration in late endosomal/lysosomal trafficking results in areduction of skin pigmentation.

[0231] By the phrase “reducing skin pigmentation” is meant a detectabledecrease in the amount of melanin in the skin, preferably causing alightening of the skin as a result of a lowering of the amount ofmelanin synthesized de novo. The term “lowering” preferably refers to aabout 10% to about a 100% decrease in the amount of melanin synthesizedde novo. More preferably, the term “lowering” refers to about a 25% toabout a 100% decrease in the amount of melanin synthesized de novo. Mostpreferably, the term “lowering” refers to about a 50 to about a 100%decrease in the amount of melanin synthesized de novo. This lowering ofmelanin synthesized de novo is preferably visually distinguishable tothe naked eye, i.e., would not require the aid of a microscope or othersuch means to detect its occurrence.

[0232] The invention also provides for a reduction in skin pigmentationby contacting the skin topically with an effective amount of a compoundthat alters late endosomal/lysosomal trafficking in the skin. Usefulcompounds for these methods of the invention include those disclosedabove.

[0233] 4. Pharmaceutical Applications

[0234] For pharmaceutical uses, it is preferred that the compound thataffects or mimics P protein function or that inhibits lateendosomal/lysosomal trafficking is part of a pharmaceutical composition.Pharmaceutical compositions, comprising an effective amount of such acompound in a pharmaceutically acceptable carrier, can be administeredto a patient, person, or animal having a disease, disorder, or conditionwhich is of a type that produces, or overproduces, melanin.

[0235] The amount of compound which will be effective in the treatmentof a particular disease, disorder, or condition will depend on thenature of the disease, disorder, or condition, and can be determined bystandard clinical techniques. Where possible, it is desirable todetermine in vitro the cytotoxicity of the compound to the tissue typeto be treated, and then in a useful animal model system prior to testingand use in humans.

[0236] The compound can be administered for the reduction or increase ofmelanin synthesis by any means that results in contact of the activeagent with its site of action in the body of a mammal. The compounds canbe administered by any conventional means available for use inconjunction with pharmaceuticals, either as individual therapeuticagents or in a combination of therapeutic agents. Each can beadministered alone, but is preferably administered with a pharmaceuticalcarrier selected on the basis of the chosen route of administration andstandard pharmaceutical practice. The pharmaceutical compositions of theinvention can be adapted for oral, parenteral, rectal, and preferablytopical, administration, and can be in unit dosage form, in a mannerwell known to those skilled in the pharmaceutical art. Parenteraladministration includes but is not limited to, injection subcutaneously,intravenously, intraperitoneally or intramuscularly. However, topicalapplication is preferred.

[0237] 5. Cosmetic Applications

[0238] In addition to pharmaceutical uses, the methods of the currentinvention are useful for cosmetic purposes. Cosmetic applications formethods of the present invention include the topical application ofcompositions containing one or more compounds to enhance or otherwisealter the visual appearance of skin or hair. Occurrences in the skin orhair of noticeable but undesired pigmentation as a result of melaninproduction, overproduction or underproduction can be treated using themethods of the present invention.

[0239] 6. Endpoints and Dosages

[0240] An effective dosage and treatment protocol can be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Animal studies,preferably mammalian studies, are commonly used to determine the maximaltolerable dose, or MTD, of a bioactive agent per kilogram weight. Thoseskilled in the art can extrapolate doses for efficacy and avoidance oftoxicity to other species, including humans.

[0241] Before human studies of efficacy are undertaken, Phase I clinicalstudies in normal subjects can help establish safe doses. Numerousfactors can be taken into consideration by a clinician when determiningan optimal dosage for a given subject. Primary among these is thetoxicity and half-life of the chosen compound that affects or mimics Pprotein function or that inhibits late endosomal/lysosomal trafficking.Additional factors include the size of the patient, the age of thepatient, the general condition of the patient, the particular disease,condition, or disorder being treated, the severity of the disease,condition, or disorder being treated, the presence of other drugs in thepatient, the effect desired, and the like. The trial dosages would bechosen after consideration of the results of animal studies and theclinical literature.

[0242] One of ordinary skill in the art will appreciate that theendpoint chosen in a particular case will vary according to the disease,condition, or disorder being treated, the outcome desired by thepatient, subject, or treating physician, and other factors. Where thecomposition is being used to lighten or darken skin color such as, forexample, to reverse hyperpigmentation caused by, for example,inflammation or diseases such as melasma, or to lighten or darken haircolor, any one of a number of endpoints can be chosen. For example,endpoints can be defined subjectively such as, for example, when thesubject is simply “satisfied” with the results of the treatment. Forpharmacological compositions, the endpoint can be determined by thepatient's, or the treating physicians, satisfaction with the results ofthe treatment. Alternatively, endpoints can be defined objectively. Forexample, the patients or subjects skin or hair in the treated area canbe compared to a color chart. Treatment is terminated when the color ofthe skin or hair in the treated area is similar in appearance to a coloron the chart. Alternatively, the reflectance of the treated skin or haircan be measured, and treatment can be terminated when the treated skinor hair attains a specified reflectance. Alternatively, the melanincontent of the treated hair or skin can be measured. Treatment can beterminated when the melanin content of the treated hair or skin reachesa specified value. Melanin content can be determined in any way known tothe art, including by histological methods, with or without enhancementby stains for melanin.

[0243] 7. Methods of Administration

[0244] The compound that affects or mimics P protein function or thatinhibits late endosomal/lysosomal trafficking or that inhibits ATPases(i.e., the active agent or ingredient) can be administered topically,e.g., as patches, ointments, creams, gels, lotions, solutions, ortransdermal administration. The compound can also be administered orallyin solid or semi-solid dosage forms, such as hard or soft-gelatincapsules, tablets, or powders, or in liquid dosage forms, such aselixirs, syrups, or suspensions. Additionally, the compound can also beadministered parenterally, in sterile liquid dosage forms or insuppository form.

[0245] Because in vivo use is contemplated, the composition ispreferably of high purity and substantially free of potentially harmfulcontaminants, e.g., at least National Food (NF) grade, generally atleast analytical grade, and preferably at least pharmaceutical grade. Tothe extent that a given compound must be synthesized prior to use, suchsynthesis or subsequent purification shall preferably result in aproduct that is substantially free of any potentially contaminatingtoxic agents that may have been used during the synthesis orpurification procedures.

[0246] Useful pharmaceutical dosage forms for administration ofcompounds that affect or mimic P protein function or that inhibit lateendosomal/lysosomal trafficking or that inhibit ATPases are describedbelow.

[0247] The pharmaceutical compositions can be applied directly to theskin. Alternatively, they can be delivered by various transdermal drugdelivery systems, such as transdermal patches as known in the art. Forexample, for topical administration, the active ingredient can beformulated in a solution, gel, lotion, ointment, cream, suspension,paste, liniment, powder, tincture, aerosol, patch, or the like in apharmaceutically or cosmetically acceptable form by methods well knownin the art. The composition can be any of a variety of forms common inthe pharmaceutical or cosmetic arts for topical application to animalsor humans, including solutions, lotions, sprays, creams, ointments,salves, gels, etc., as described below. Preferred agents are those thatare viscous enough to remain on the treated area, those that do notreadily evaporate, and/or those that are easily removed by rinsing withwater, optionally with the aid of soaps, cleansers and/or shampoos.Actual methods for preparing topical formulations are known or apparentto those skilled in the art, and are described in detail in Remington'sPharmaceutical Sciences, 1990 (supra); and Pharmaceutical Dosage Formsand Drug Delivery Systems, 6th ed., Williams & Wilkins (1995).

[0248] In order to enhance the percutaneous absorption of the activeingredients, one or more of a number of agents can be added in thetopical formulations including, but not limited to, dimethylsulfoxide,dimethylacetamide, dimethylformamide, surfactants, azone, alcohol,acetone, propylene glycol and polyethylene glycol. In addition, physicalmethods can also be used to enhance transdermal penetration such as,e.g., by iontophoresis or sonophoresis. Alternatively, or in addition,liposomes may be employed.

[0249] A topically applied composition of the invention contains apharmaceutically effective agent that affects or mimics P proteinfunction or that inhibits late endosomal/lysosomal trafficking or thatinhibits ATPases as described herein, and those ingredients as arenecessary for use as a carrier, such as an emulsion, a cream, anointment, an ophthalmic ointment, an aqueous solution, a lotion or anaerosol. Non-limiting examples of such carriers are described in moredetail below and may be found in International Pat. Publication WO00/62742, published Oct. 26, 2000, U.S. Pat. No. 5,691,380 to Mason etal., issued on Nov. 25, 1997 and U.S. Pat. No. 5,968,528 to Deckner etal., issued on Oct. 19, 1999, U.S. Pat. No. 4,139,619 to Chidsey, III,issued on Feb. 13, 1979 and U.S. Pat. No. 4,684,635 to Orentreich etal., issued on Aug. 4, 1987 which are incorporated herein by reference.Suitable pharmaceutical carriers are further described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.(1990) a standard reference text in this field.

[0250] The pharmaceutical compositions of the invention may also includeoptional components. Such optional components should be suitable forapplication to keratinous tissue, that is, when incorporated into thecomposition, they are suitable for use in contact with human keratinoustissue without undue toxicity, incompatibility, instability, allergicresponse, and the like within the scope of sound medical judgment. Inaddition, such optional components are useful provided that they do notunacceptably alter the benefits of the active compounds of theinvention. The CTFA Cosmetic Ingredient Handbook, Second Edition (1992)describes a wide variety of non-limiting cosmetic and pharmaceuticalingredients commonly used in the skin care industry, which are suitablefor use in the compositions of the present invention. Examples of theseingredient classes include: abrasives, absorbents, aesthetic componentssuch as fragrances, pigents, colorings/colorants, essential oils, skinsensates, astringents, etc. (e.g., clove oil, menthol, camphor,eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate),anti-acne agents, anti-caking agents, antifoaming agents, antimicrobialagents (e.g., iodopropyl butylcarbamate), antioxidants, binders,biological additives, buffering agents, bulking agents, chelatingagents, chemical additives, colorants, cosmetic astringents, cosmeticbiocides, denaturants, drug astringents, external analgesics, filmformers or materials, e.g., polymers, for aiding the film-formingproperties and substantivity of the composition (e.g., copolymer ofeicosene and vinyl pyrrolidone), opacifying agents, pH adjusters,propellants, reducing agents, sequestrants, skin-conditioning agents(e.g., humectants, including miscellaneous and occlusive), skin soothingand/or healing agents (e.g., panthenol and derivatives (e.g., ethylpanthenol), aloe vera, pantothenic acid and its derivatives, allantoin,bisabolol, and dipotassium glycyffhizinate), skin treating agents,thickeners, and vitamins and derivatives thereof.

[0251] In addition to the pharmaceutically effective amount of an agentdisclosed herein, the topical compositions of the present invention alsocomprise a dermatologically acceptable carrier. The phrase“dermatologically acceptable carrier”, as used herein, means that thecarrier is suitable for topical application to the skin, i.e.,keratinous tissue, has good aesthetic properties, is compatible with theactive agents of the present invention and any other components, andwill not cause any safety or toxicity concerns. A safe and effectiveamount of carrier is from about 50% to about 99.99%, preferably fromabout 80% to about 99.9%, more preferably from about 90% to about 98%,and most preferably from about 90% to about 95% of the composition.

[0252] The carrier utilized in the compositions of the invention can bein a wide variety of forms. These include emulsion carriers, including,but not limited to, oil-in-water, water-in-oil, water-in-oil-in-water,and oil-in-water-in-silicone emulsions, a cream, an ointment, anophthalmic ointment, an aqueous solution, a lotion or an aerosol. Aswill be understood by the skilled artisan, a given component willdistribute primarily into either the water or oil/silicone phase,depending on the water solubility/dispersibility of the component in thecomposition.

[0253] Emulsions according to the present invention generally contain apharmaceutically effective amount of an agent disclosed herein and alipid or oil. Lipids and oils may be derived from animals, plants, orpetroleum and may be natural or synthetic (i.e., man-made). Preferredemulsions also contain a humectant, such as glycerin. Emulsions willpreferably further contain from about 1% to about 10%, more preferablyfrom about 2% to about 5%, of an emulsifier, based on the weight of thecarrier. Emulsifiers may be nonionic, anionic or cationic. Suitableemulsifiers are described in, for example, U.S. Pat. No. 3,755,560,issued to Dickert, et al. Aug. 28, 1973; U.S. Pat. No. 4,421,769, issuedto Dixon, et al.. Dec. 20, 1983; and McCutcheon's Detergents andEmulsifiers, North American Edition, pages 317-324 (1986).

[0254] The emulsion may also contain an anti-foaming agent to minimizefoaming upon application to the keratinous tissue. Anti-foaming agentsinclude high molecular weight silicones and other materials well knownin the art for such use.

[0255] Suitable emulsions may have a wide range of viscosities,depending on the desired product form. Exemplary low viscosityemulsions, which are preferred, have a viscosity of about 50 centistokesor less, more preferably about 10 centistokes or less, most preferablyabout 5 centistokes or less. The emulsion may also contain ananti-foaming agent to minimize foaming upon application to thekeratinous tissue. Anti-foaming agents include high molecular weightsilicones and other materials well known in the art for such use.

[0256] One type of emulsion is a water-in-silicone emulsion.Water-in-silicone emulsions contain a continuous silicone phase and adispersed aqueous phase. Preferred water-in-silicone emulsions of thepresent invention comprise from about 1% to about 60%, preferably fromabout 5% to about 40%, more preferably from about 10% to about 20%, byweight of a continuous silicone phase. The continuous silicone phaseexists as an external phase that contains or surrounds the discontinuousaqueous phase described hereinafter.

[0257] The continuous silicone phase may contain a polyorganosiloxaneoil. A preferred water-in-silicone emulsion system is formulated toprovide an oxidatively stable vehicle for delivery of a pharmaceuticallyeffective amount of an agent disclosed herein. The continuous siliconephase of these preferred emulsions comprises between about 50% and about99.9% by weight of organopolysiloxane oil and less than about 50% byweight of a non-silicone oil. In an especially preferred embodiment, thecontinuous silicone phase comprises at least about 50%, preferably fromabout 60% to about 99.9%, more preferably from about 70% to about 99.9%,and even more preferably from about 80% to about 99.9%,polyorganosiloxane oil by weight of the continuous silicone phase, andup to about 50% non-silicone oils, preferably less about 40%, morepreferably less than about 30%, even more preferably less than about10%, and most preferably less than about 2%, by weight of the continuoussilicone phase. These useful emulsion systems may provide more oxidativestability over extended periods of time than comparable water-in-oilemulsions containing lower concentrations of the polyorganosiloxane oil.Concentrations of non-silicone oils in the continuous silicone phase areminimized or avoided altogether so as to possibly further enhanceoxidative stability of the active compound of the invention in thecompositions. Water-in-silicone emulsions of this type are described inU.S. Pat. No. 5,691,380 to Mason et al., issued Nov. 25, 1997.

[0258] The organopolysiloxane oil for use in the composition may bevolatile, non-volatile, or a mixture of volatile and non-volatilesilicones. The term “nonvolatile” as used in this context refers tothose silicones that are liquid under ambient conditions and have aflash point (under one atmospheric of pressure) of or greater than about100 degrees Celsius. The term “volatile” as used in this context refersto all other silicone oils. Suitable organopolysiloxanes can be selectedfrom a wide variety of silicones spanning a broad range of volatilitiesand viscosities. Examples of suitable organopolysiloxane oils includepolyalkylsiloxanes, cyclic polyalkylsiloxanes, andpolyalkylarylsiloxanes, which are known to those skilled in the art andcommercially available.

[0259] The continuous silicone phase may contain one or morenon-silicone oils. Concentrations of non-silicone oils in the continuoussilicone phase are preferably minimized or avoided altogether so as tofurther enhance oxidative stability of the pharmaceutically effectiveagent in the compositions. Suitable non-silicone oils have a meltingpoint of about 25° C. or less under about one atmosphere of pressure.Examples of non-silicone oils suitable for use in the continuoussilicone phase are those well known in the chemical arts in topicalpersonal care products in the form of water-in-oil emulsions, e.g.mineral oil, vegetable oils, synthetic oils. semisynthetic oils, etc.

[0260] Useful topical compositions of the present invention comprisefrom about 30% to about 90%, more preferably from about 50% to about85%, and most preferably from about 70% to about 80% of a dispersedaqueous phase. In emulsion technology, the term “dispersed phase” is aterm well-known to one skilled in the art which means that the phaseexists as small particles or droplets that are suspended in andsurrounded by a continuous phase. The dispersed phase is also known asthe internal or discontinuous phase. The dispersed aqueous phase is adispersion of small aqueous particles or droplets suspended in andsurrounded by the continuous silicone phase described hereinbefore. Theaqueous phase can be water, or a combination of water and one or morewater soluble or dispersible ingredients. Nonlimiting examples of suchoptional ingredients include thickeners, acids, bases, salts, chelants,gums, water-soluble or dispersible alcohols and polyols, buffers,preservatives, sunscreening agents, colorings, and the like.

[0261] The topical compositions of the present invention typicallycomprise from about 25% to about 90%, preferably from about 40% to about80%, more preferably from about 60% to about 80%, water in the dispersedaqueous phase by weight of the composition.

[0262] The water-in-silicone emulsions of the present inventionpreferably comprise an emulsifier. In a preferred embodiment, thecomposition contains from about 0.1% to about 10% emulsifier, morepreferably from about 0.5% to about 7.5%, most preferably from about 1%to about 5%, emulsifier by weight of the composition. The emulsifierhelps disperse and suspend the aqueous phase within the continuoussilicone phase.

[0263] A wide variety of emulsifying agents can be employed herein toform the preferred water-in-silicone emulsion. Known or conventionalemulsifying agents can be used in the composition, provided that theselected emulsifying agent is chemically and physically compatible withessential components of the composition, and provides the desireddispersion characteristics. Suitable emulsifiers include siliconeemulsifiers, e.g., organically modified organopolysiloxanes, also knownto those skilled in the art as silicone surfactants,non-silicon-containing emulsifiers, and mixtures thereof, known by thoseskilled in the art for use in topical personal care products.

[0264] Useful emulsifiers include a wide variety of siliconeemulsifiers. These silicone emulsifiers are typically organicallymodified organopolysiloxanes, also known to those skilled in the art assilicone surfactants. Suitable emulsifiers are described, for example,in McCutcheon's, Detergents and Emulsifiers, North American Edition(1986), published by Allured Publishing Corporation; U.S. Pat. No.5,011,681 to Ciotti et al., issued Apr. 30, 1991; U.S. Pat. No.4,421,769 to Dixon et al., issued Dec. 20, 1983; and U.S. Pat. No.3,755,560 to Dickert et al., issued Aug. 28, 1973.

[0265] Other preferred topical carriers include oil-in-water emulsions,having a continuous aqueous phase and a hydrophobic, water-insolublephase (“oil phase”) dispersed therein. Examples of suitable carrierscomprising oil-in-water emulsions are described in U.S. Pat. No.5,073,371 to Turner, D. J. et al., issued Dec. 17, 1991, and U.S. Pat.No. 5,073,372, to Turner, D. J. et al., issued Dec. 17, 1991. Anespecially preferred oil-in-water emulsion, containing a structuringagent, hydrophilic surfactant and water, is described in detailhereinafter.

[0266] A preferred oil-in-water emulsion comprises a structuring agentto assist in the formation of a liquid crystalline gel networkstructure. Without being limited by theory, it is believed that thestructuring agent assists in providing rheological characteristics tothe composition which contribute to the stability of the composition.The structuring agent may also function as an emulsifier or surfactant.Preferred compositions of this invention comprise from about 0.5% toabout 20%, more preferably from about 1% to about 10%, most preferablyfrom about 1% to about 5%, by weight of the composition, of astructuring agent. The preferred structuring agents of the presentinvention are selected from the group consisting of stearic acid,palmitic acid, stearyl alcohol, cetyl alcohol, behenyl alcohol, stearic;acid, palmitic acid, the polyethylene glycol ether of stearyl alcoholhaving an average of about 1 to about 21 ethylene oxide units, thepolyethylene glycol ether of cetyl alcohol having an average of about 1to about 5 ethylene oxide units, and mixtures thereof.

[0267] The preferred oil-in-water emulsions comprise from about 0.05% toabout 10%, preferably from about 1% to about 6%, and more preferablyfrom about 1% to about 3% of at least one hydrophilic surfactant whichcan disperse the hydrophobic materials in the water phase (percentagesby weight of the topical carrier). The surfactant, at a minimum, must behydrophilic enough to disperse in water. Suitable surfactants includeany of a wide variety of known cationic, anionic, zwitterionic, andamphoteric surfactants. See, McCutcheon's. Detergents and Emulsifiers,North American Edition (1986), published by Allured PublishingCorporation; U.S. Pat. No. 5,011,681 to Ciotti et al., issued Apr. 30,1991; U.S. Pat. No. 4,421,769 to Dixon et al. issued to Dec. 20, 1983;and U.S. Pat. No. 3,755,560. The exact surfactant chosen depends uponthe pH of the composition and the other components present. Preferredare cationic surfactants, especially dialkyl quaternary ammoniumcompounds, examples of which are described in U.S. Pat. No. 5,151,209 toMcCall et al. issued to Sep. 29, 1992; U.S. Pat. No. 5,151,210 to Steuriet al.. issued to Sep. 29, 1992; U.S. Pat. Nos. 5,120,532; U.S. Pat. No.4,387,090; U.S. Pat. No. 3,155,591; U.S. Pat. No. 3,929,678; U.S. Pat.No. 3,959,461; McCutcheon's, Detergents & Emulsifiers (North Americanedition 1979) M.C. Publishing Co.; and Schwartz, et al., Surface ActiveAgents, Their chemistry and Technology, New York: IntersciencePublishers, 1949.

[0268] Alternatively, other useful cationic emulsifiers includeamino-amides. Nonlimiting examples of these cationic emulsifiers includestearamidopropyl PG-dimonium chloride phosphate, behenamidopropyl PGdimonium chloride, stearamidopropyl ethyldimonium ethosulfate,stearamidopropyl dimethyl (myristyl acetate) ammonium chloride,stearamidopropyl dimethyl cetearyl ammonium tosylate, stearamidopropyldirnethyl ammonium chloride, stearamidopropyl dimethyl ammonium lactate,and mixtures thereof.

[0269] A wide variety of anionic surfactants are also useful herein.See, e.g., U.S. Pat. No. 3,929,678, to Laughlin et al., issued Dec. 30,1975. In addition, amphoteric and zwitterionic surfactants are alsouseful herein.

[0270] The preferred oil-in-water emulsion comprises from about 25% toabout 98%, preferably from about 65% to about 95%, more preferably fromabout 70% to about 90% water by weight of the topical carrier.

[0271] The hydrophobic phase is dispersed in the continuous aqueousphase. The hydrophobic phase may contain water insoluble or partiallysoluble materials such as are known in the art, including but notlimited to the silicones described herein in reference tosilicone-in-water emulsions, and other oils and lipids such as describedabove in reference to emulsions.

[0272] The topical compositions of the subject invention, including butnot limited to lotions and creams, may comprise a dermatologicallyacceptable emollient. Such compositions preferably contain from about 2%to about 50% of the emollient. As used herein, “emollient” refers to amaterial useful for the prevention or relief of dryness, as well as forthe protection of the skin. A wide variety of suitable emollients areknown and may be used herein. See, e.g., Sagarin, Cosmetics, Science andTechnology, 2nd Edition, Vol. 1, pp. 3243 (1972), which containsnumerous examples of materials suitable as an emollient. A preferredemollient is glycerin. Glycerin is preferably used in an amount of fromor about 0.001 to or about 20%, more preferably from or about 0.01 to orabout 10%, most preferably from or about 0.1 to or about 5%, e.g., 3%.

[0273] Lotions and creams according to the present invention generallycomprise a solution carrier system and one or more emollients. Lotionstypically comprise from about 1% to about 20%, preferably from about 5%to about 10% of emollient; from about 50% to about 90%, preferably fromabout 60% to about 80% water; and a pharmaceutically effective amount ofan agent described herein. A cream typically comprises from about 5% toabout 50%, preferably from about 10% to about 20% of emollient; fromabout 45% to about 85%, preferably from about 50% to about 75% water;and a pharmaceutically effective amount of an agent described herein.

[0274] Ointments of the present invention may comprise a simple carrierbase of animal or vegetable oils or semi-solid hydrocarbons(oleaginous); absorption ointment bases which absorb water to formemulsions; or water soluble carriers, e.g., a water soluble solutioncarrier. Ointments may further comprise a thickening agent, such asdescribed in Sagarin, Cosmetics, Science and Technology, 2nd Edition,Vol. 1, pp. 72-73 (1972), incorporated herein by reference, and/or anemollient. For example, an ointment may comprise from about 2% to about10% of an emollient; from about 0.1% to about 2% of a thickening agent;and a pharmaceutically effective amount of an agent described herein.

[0275] By way of non-limiting example, 1000 g of topical cream isprepared from the following types and amounts of ingredients: apharmaceutically effective amount of an agent disclosed herein, tegacidregular (150 g) (a self-emulsifying glyceryl monostearate fromGoldschmidt Chemical Corporation, New York, N.Y.), polysorbate 80 (50g), spermaceti (100 g), propylene glycol (50 g), methylparaben (1 g),and deionized water in sufficient quantity to reach 1000 gm. The tegacidand spermaceti are melted together at a temperature of 70-80° C. Themethylparaben is dissolved in about 500 g of water and the propyleneglycol, polysorbate 80, and6-amino-1,2-dihydro-1-hydroxy-2-imino-4-piperidinopyrimidine free baseare added in turn, maintaining a temperature of 75-80° C. Themethylparaben mixture is added slowly to the tegacid and spermacetimelt, with constant stirring. The addition is continued for at least 30minutes with additional stirring until the temperature has dropped to40-45° C. Finally, sufficient water is added to bring the final weightto 1000 g and the preparation stirred to maintain homogeneity untilcooled and congealed.

[0276] By way of non-limiting example, 1000 g of a topical ointment isprepared from the following types and amounts of ingredients: apharmaceutically effective amount of an agent disclosed herein, zincoxide (50 g), calamine (50 g), liquid petrolatum (heavy) (250 g), woolfat (200 g), and enough white petrolatum to reach 1000 g. Briefly, thewhite petrolatum and wool fat are melted and 100 g of liquid petrolatumadded thereto. The pharmaceutically effective amount of an agentdisclosed herein, zinc oxide, and calamine are added to the remainingliquid petrolatum and the mixture milled until the powders are finelydivided and uniformly dispersed. The mixture is stirred into the whitepetrolatum, melted and cooled with stirring until the ointment congeals.

[0277] By way of non-limiting example, 1000 g of an ointment, e.g., anophthalmic ointment, containing a pharmaceutically effective amount ofan agent disclosed herein is prepared from the following types andamounts of ingredients: a pharmaceutically effective amount of an agentdisclosed herein, light liquid petrolatum (250 g), wool fat (200 g), andenough white petrolatum to reach 1000 g. Briefly, the pharmaceuticallyeffective amount of an agent disclosed herein is finely divided andadded to the light liquid petrolatum. The wool fat and white petrolatumare melted together, strained, and the temperature adjusted to 45-50° C.The liquid petrolatum slurry is added, and the ointment stirred untilcongealed.

[0278] By way of non-limiting example, 1000 ml of an aqueous solutioncontaining a pharmaceutically effective amount of an agent disclosedherein is prepared from the following types and amounts of ingredients:a pharmaceutically effective amount of an agent disclosed herein,polyethylene glycol 4000 (120 g) myristyl-gamma-picolinium chloride (0.2g), polyvinylpyrrolidone (1 g), and enough deionized water to reach 1000milliliters. Briefly, the ingredients are dissolved in the water and theresulting solution is sterilized by filtration.

[0279] By way of non-limiting example, 1000 g of lotion containing apharmaceutically effective amount of an agent disclosed herein isprepared from the following types and amounts of ingredients: apharmaceutically effective amount of an agent disclosed herein, N-methylpyrolidone (40 g), and enough propylene glycol to reach 1000 g.

[0280] By way of non-limiting example, an aerosol containing apharmaceutically effective amount of an agent disclosed herein isprepared from the following types and amounts of materials: apharmaceutically effective amount of an agent disclosed herein, absolutealcohol (4.37 g), Dichlorodifluoroethane (1.43 g) anddichlorotetrafluoroethane (5.70 g). Briefly, the pharmaceuticallyeffective amount of an agent disclosed herein is dissolved in theabsolute alcohol and the resulting solution filtered to remove particlesand lint. This solution is chilled to about minus 30° C. Then, to thisis added the chilled mixture of dichlorodifluoromethane anddichlorotetrafluoroethane.

[0281] For oral administration, Gelatin capsules or liquid-filled softgelatin capsules can contain the active ingredient and powdered orliquid carriers, such as lactose, lecithin starch, cellulosederivatives, magnesium stearate, stearic acid, and the like. Similardiluents can be used to make compressed tablets. Both tablets andcapsules can be manufactured as sustained release products to providefor continuous release of medication over a period of hours. Compressedtablets can be sugar-coated or film-coated to mask any unpleasant tasteand to protect the tablet from the atmosphere, or enteric-coated forselective, targeted disintegration in the gastrointestinal tract. Liquiddosage forms for oral administration can contain coloring and/orflavoring to increase patient acceptance.

[0282] In general, sterile water, oil, saline, aqueous dextrose(glucose), polysorbate and related sugar solutions and glycols such aspropylene glycol or polyethylene glycols, are suitable carriers forparenteral solutions. Solutions or emulsions for parenteraladministration preferably contain about 5-15% polysorbate 80 orlecithin, suitable stabilizing agents and, if necessary, buffersubstances. Antioxidizing agents such as, but not limited to, sodiumbisulfite, sodium sulfite, or ascorbic acid, either alone or combined,are suitable stabilizing agents. Also useful are citric acid and itssalts, and sodium EDTA. In addition, parenteral solutions can containpreservatives including, but not limited to, benzalkonium chloride,methyl- or propyl-paraben, and chlorobutanol.

[0283] As will be understood by those in the art, the compositions andpharmaceutical compositions of the invention may be provided in the formof a kit. Kits of the invention comprise one or more specificcompositions and/or pharmaceutical compositions of the invention thatmimic and/or affect P protein function, inhibit late endosomal/lysosomaltrafficking, or inhibit ATPases. Optionally, the kit further containsprinted instructions as a label or package insert directing the use ofsuch reagents to modulate skin pigmentation, i.e., to either lighten ordarken skin as appropriate to the particular included composition. Thesecompounds are provided in a container designed to prevent contamination,minimize evaporation or drying of the composition, etc. The compoundsmay or may not be provided in a preset unit dose or usage amount.

[0284] The invention having been described, the following examples areoffered by way of illustration and not limitation.

EXAMPLES Example 1

[0285] Targeting Function Screen

[0286] In this example, the effect of P protein on cellular targeting oftyrosinase was investigated. This function was then exploited in ascreen for compounds that inhibit the activity of P protein.

[0287] Melan-a cells (a/a, PIP), an immortalized melanocyte line derivedfrom C57BL1 6J mice wild type at the p locus (Bennett et al., 1987, Int.J. Cancer 39:414-418), were maintained in culture in Dulbecco'smodification of Eagle's medium (DME). Melan-p1 melanocytes from micelacking all p gene transcripts due to the presence of overlappingdeletions (a/a, p^(cp)/p^(25H)) (Sviderskaya et al., 1997, J. Invest.Dermatol. 108:30-34) were maintained in Ham's F10 medium. Both mediawere supplemented with 10% fetal calf serum, 5% sodium pyruvate, 5%glutamate, 5 units/ml penicillin, 5 μg/ml streptomycin, 1% non-essentialamino acids and 200 nM 12-0-tetradecanoyl phorbol 13-acetate. Inaddition, 200 pM cholera toxin was added to the melan-p1 cells.

[0288] Cells were maintained in the appropriate media, which was thenreplaced with tyrosine deficient DME medium (DME-D) supplemented witheither 0.03 mM tyrosine for low tyrosine conditions or 0.3 mM tyrosinefor high tyrosine conditions (Bennett, D.C. et al., 1987, Int. J. Cancer39:414-418), (Sviderskaya et al., J. Invest Dermatol. 108:30-34).Aliquots of culture medium were withdrawn, dialyzed against 0.1 M sodiumphosphate buffer, pH 6.8, and analyzed for tyrosinase activity using aradiometric tyrosine hydroxylase assay (Orlow, S. J. et al., 1990, J.Invest. Dermatol 94:461-64).

[0289] For treatment with test compounds, cultured melan-a melanocyteswere incubated for 48 hours in the presence of low tyrosine in themedium as above but in the presence of benztropine (10 micromolar finalconcentration), or imipramine (10 micromolar final concentration), ornitroquipazine, (30 micromolar final concentration), or left untreated.Incubation media were assayed for tyrosinase activity, as above.

[0290] Increasing tyrosinase activity in the media removed from melan-pcell cultures grown in the presence of low tyrosine indicates that thesecells secrete relatively large amounts of tyrosinase into theirincubation media (FIG. 1). By contrast, melan-a cells, which representwild type melanocytes, secrete significantly less tyrosinase into themedia (FIG. 1). While culture in the presence of excess tyrosine hadlittle effect on melan-a cells, the amount of enzyme secreted bymelan-p1 cells was reduced. As predicted above, tyrosine appears topartially correct the misrouting of tyrosinase in melan-p1 cells.

[0291] Treatment with benztropine did not alter the levels of tyrosinaseactivity secreted to the incubation medium of melan-a cells (FIG. 2).Treatment with either imipramine or nitroquipazine significantlyincreased the levels of tyrosinase activity found in the cells'incubation medium (FIG. 2).

[0292] Melan-a cells are melanocytes derived from wild type mice. Theyhave fully functional P protein and tyrosinase, and produce melanin.Melan-p cells, however, are derived from p-null mice having a deletionof the entire p gene coding sequence. Thus, they produce no P protein.Consequently, melan-p cells have lower tyrosinase activity and make lessmelanin than melan-a cells.

[0293] This example, which can be performed with any type of melanogeniccell, demonstrates that melanocytes lacking P protein function secretesignificantly more tyrosinase into their growth or incubation mediumthan do melanocytes with normal P protein function. This result isobtained either when the cells are genetically altered to reduce oreliminate P protein function, as in melan-p cells (FIG. 1), or when thecells are treated with a compound that inhibits P protein function, suchas imipramine (FIG. 2B).

Example 2

[0294] Tyrosinase Activity Screen

[0295] In this example, the effect of P protein on the measurableenzymatic activity of tyrosinase from cells genetically engineered toexpress tyrosinase was investigated. Any melanogenic cell type thatexpresses both P protein and tyrosinase, or any cell type made toexpress both P protein and tyrosinase, can be substituted. This functionwas then exploited in a screen for compounds that inhibit the functionof P protein.

[0296] Cultured melan-a melanocytes, as described above in Example 1,were incubated for 48 hours in the presence of benztropine (10micromolar final concentration), or imipramine (10 micromolar finalconcentration), or nitroquipazine (30 micromolar final concentration),or left untreated. Cells were washed and extracted with 50 mM Tris-HCl(pH 7.4), 2 mM EDTA, 150 mM NaCl and 1% Triton X-100. Cell extracts wereanalyzed for tyrosinase activity using a radiometric tyrosinehydroxylase assay (Orlow, S. J. et al., 1990, supra).

[0297] Expression vectors were constructed to express P protein andtyrosinase genes in cultured cells. Specifically, the coding sequencefor tyrosinase was removed as a HindIII-EcoRI fragment from clone TYBS(Yokohama et al., 1990, Nucl. Acids. Res. 18:7293-7298) and cloned intothe HindIII/EcoRI sites of pcDNA I/amp (Invitrogen, Calif.). Codingsequence for the P protein was removed as a BamHI-EcoRI fragment fromMC2701 (Gardner et al., 1992, supra) and cloned into the BamHI/EcoRVsites of pcDNA3 and pcDNA3.1/V5/His-TOPO (Invitrogen, Calif.). COS cellswere transfected with the pcDNA1-based plasmids and FuGENE™ 6 (RocheMolecular Biochemicals, Indianapolis, Ind.) as transfection agents for48 hours. Cells were transformed with: (i) the vector alone; (ii) thevector carrying a tyrosinase-encoding gene; (iii) the vector carrying aP protein-encoding gene; or (iv) vectors carrying a tyrosinase-encodinggene and a P protein-encoding gene. Transformed cells were washed andextracted as above. Tyrosinase activity was then measured as above.Tyrosinase assays were performed on 60 micrograms of cell protein.

[0298] COS cells transfected with a vector carrying atyrosinase-encoding gene, or with vectors carrying a tyrosinase-encodinggene and a P protein-encoding gene as above, were treated withbenztropine, or imipramine, or nitroquipazine, or left untreated, asabove, and cell extracts were then prepared as above. The tyrosinaseactivity of cell extracts was determined as above.

[0299] As shown in FIG. 2A, extracts from melan-a cells treated withbenztropine or nitroquipazine had greater tyrosinase activities thanuntreated cells. Extracts from cells treated with imipramine had lesstyrosinase activity than untreated cells.

[0300] As shown in FIG. 3, extracts from COS cells transfected with thevector alone (V+V) or with the vector carrying the P protein-encodinggene (V+P) did not exhibit measurable tyrosinase activity. Extracts fromcells transfected with the vector carrying the tyrosinase-encoding gene(V+T) had measurable tyrosinase activity, while extracts from cellstransfected with the vectors carrying the tyrosinase-encoding gene andthe P protein-encoding gene (T+P) had tyrosinase activity approximatelyfour fold greater than the tyrosinase activity found in extracts ofcells transfected with the vector carrying the tyrosinase-encoding genealone (V+T).

[0301]FIG. 4 shows the separate effects of three compounds on P proteinfunction. Nitroquipazine (4) caused extracts from tyrosinase-expressingCOS cells to exhibit lower tyrosinase activity, regardless of whetherthe cells were expressing the P protein. Benztropine (2) did not have anappreciable effect on tyrosinase activity in these extracts. Imipramine(3) dramatically reduced the tyrosinase activity of cells expressingboth P protein and tyrosinase, but had very little effect on cellsexpressing only tyrosinase.

[0302] This example illuminates the relationship between P proteinfunction and tyrosinase activity in cell extracts. Melanocytes thatexpress P protein can be made to mimic cells that lack P proteinfunction through the use of compounds that inhibit P protein function.Melan-a cells are wild type for the P protein-encoding gene. Yetextracts taken from these cells after they are treated with imipraminehave lower tyrosinase activity than untreated melan-a cells (FIG. 2). Incontrast, extracts from cells treated with benztropine or nitroquipazinehave higher tyrosinase activity than untreated cells (FIG. 2).

[0303] COS cells are derived from monkey kidney cells. Normally, they donot express tyrosinase or P protein. This example demonstrates that bytransfecting COS cells with a tyrosinase-encoding gene and a P proteinencoding gene, one can produce what might be considered an “artificialmelanocyte.” These cells express active tyrosinase and P protein (FIG.3), and even produce melanin. Cotransfection of COS cells with both atyrosinase-encoding gene and a P protein-encoding gene produces cellswith approximately four times more tyrosinase activity than COS cellstransfected with a tyrosinase-encoding gene alone (FIG. 3). This resultdemonstrates that P protein is expressed and active in these cellsbecause the intracellular activity of tyrosinase was increased by Pprotein expression.

[0304] Extracts from COS cells that have been transformed with both atyrosinase-encoding gene and a P protein-encoding gene and then treatedwith imipramine contained only about one third of the tyrosinaseactivity of similar cells not treated with imipramine (FIG. 4). Thetyrosinase activity of COS cells that were transfected with only atyrosinase-encoding gene and then treated with imipramine was notsignificantly different than the tyrosinase activity of extracts ofsimilar cells not treated with imipramine (FIG. 4). These resultsindicate that imipramine reduces tyrosinase activity by inhibiting Pprotein function. By contrast, benztropine did not reduce the tyrosinaseactivity of extracts of transfected COS cells, whether or not theyexpressed P protein (FIG. 4). In addition, nitroquipazine reduced thetyrosinase activity of extracts of transfected COS cells, whether or notthey expressed P protein (FIG. 4). This result indicates thatnitroquipazine is not an inhibitor of P protein function.

Example 3

[0305] Secretion of Tyrosinase in Melan-p Cells Results from Proteolysis

[0306] While we observed activity of tyrosinase in the medium, Potterfet al. (1998, Exp. Cell Res. 244:319-326) did not detect tyrosinaseprotein in the medium using αPEP7. Tyrosinase is a type I membraneprotein anchored in the membrane, and it is thus likely thatproteolysis, which leads to the clipping of the tail, is required forsecretion. The truncated protein would not be detected by αPEP7, whichis directed against the tail, although the catalytic domains wouldremain functional. We, therefore, examined the effects of a series ofprotease inhibitors on the secretion of tyrosinase by melan-a andmelan-p1 cells. E64, an epoxysuccinyl peptide and a potent inhibitor ofcysteine proteinases was found to be the most effective in reducing theamount of tyrosinase secreted into the media of melan-p1 cells (FIG.5A), thus demonstrating that secretion of tyrosinase can be inhibited byblocking the activity of cysteinyl proteases.

[0307] If proteolysis and secretion of tyrosinase were the precipitatingfactor in the misrouting of tyrosinase, then E64 should increase melaninaccumulation in melan-p1 cells. The effects of E64 were furtherinvestigated, and a potential synergy with tyrosine, which also reducedsecretion into the media, examined. A range of E64 concentrations wastested at low (0.03 mM) and high (0.3 mM) tyrosine.

[0308] At 0.03 mM tyrosine, 12.5 μM E64 lowered secretion of tyrosinaseinto the medium from 7.1% to 4.0% (FIG. 5a), whereas at higherconcentrations (25 μM), E64 was only slightly more effective (3.8%activity in media). E64 also reduced tyrosinase secretion at highertyrosine concentrations (0.3 mM), reducing the tyrosinase in the mediumfrom 6.5% to 3.9%. The higher concentration of E64 was not moreeffective. Surprisingly, E64 reduced intracellular melanin production athigh concentrations of tyrosine. Thus, despite its ability to diminishproteolysis and secretion of tyrosinase from melan-p1 cells, E64 was notable to cause tyrosinase to re-route to the melanosome and begin melaninsynthesis and deposition.

Example 4

[0309] Comparison of Ultrastructure and Distribution of Tyrosinase inMelan-a and Melan-p1 Cells

[0310] Melanocytes were seeded into Lab-Tek chamber slides (Nunc, Inc.,Naperville, Ill.) and grown to 90% confluence. Cultured melanocytes werefixed in wells with half-strength Karnovsky's fixative (Strum, et al.(1970) J. Ultrastruct Res. 31(3):323-36) in 0.2 M sodium cacodylatebuffer at pH 7.2 for 30 minutes at room temperature. Fordihydroxyphenylalanine (DOPA) (Sigma, St. Louis, Mo.) histochemistry,fixed cells were incubated in 0.1% L-DOPA twice for 2.5 hours. The cellswere washed 3 times in buffer and treated with 1.0% osmium tetroxidecontaining 1.5% potassium ferrocyanide (Strum, supra) for 30 minutes.The cells were washed, stained en bloc with 0.5% uranyl acetate for 30minutes, dehydrated, and embedded in Eponate 12. Areas of the Epon casewere cut out and mounted on Epon pegs and sectioned on an RMC MT 6000-XLultramicrotome. Ultrathin sections were stained with aqueous solutionsof uranyl acetate (2%) and lead citrate (0.3%) for 15 minutes each, andthen viewed and photographed in a JEOL JEM-IOOCX transmission electronmicroscope.

[0311] Previous studies have shown that deficiency of the P proteinresults in both ultrastructural aberrations (Moyer, 1966, Am. Zool6:43-66; Sidman and Pearlstein, 1965, Dev. Biol. 12:93-116; Orlow andBrilliant, 1999, Exp. Eye Res. 68:147-154), as well as abnormalsubcellular localization of tyrosinase (Potterf et al., 1998, supra). Inorder to investigate both features simultaneously, we determined thesubcellular architecture and the distribution of tyrosinase in melan-aand melan-p1 cells by electron microscopy with and without DOPAhistochemistry.

[0312] As reported previously (Rosemblat et al., 1998, Exp. Cell Res.239:344-352), cultured melan-a cells, wild type at the p locus,contained melanosomes that were predominantly of stage IV maturation(FIG. 6A). In contrast, p-null melan-p1 cells exhibited melanosomes thatwere predominantly stage I and II and occasionally stage III (FIG. 6B).

[0313] Upon DOPA incubation of melan-a cells, tyrosinase activity wasdemonstrated in the trans Golgi network (TGN) and in 50 nm vesicleswhich were confined to the vicinity of the Golgi apparatus (FIG. 7A).DOPA treated melan-p1 cells also demonstrated reaction product in theTGN and neighboring 50 nm vesicles (FIG. 7B). In addition, reactionproduct was present in some melan-p1 melanosomes. However, manymelanosomes, both in the cell body as well as in the dendrites, remaineddevoid of reaction product (FIG. 7B). Unlike melan-a cells (FIG. 7A),melan-p1 cells exhibited reaction product in 50 nm vesicles well outsideof the peri-nuclear Golgi area (FIG. 7B) and in close proximity to theplasma membrane (FIG. 7B) suggesting an abnormal accumulation oftyrosinase in a population of vesicles.

[0314] The lack of P protein resulted in the proliferation of smalltyrosinase-containing vesicles that were no longer limited to the areaaround the TGN. Tyrosinase was, therefore, either packaged intodifferent vesicles in the two cell lines or, alternatively, the vesicleswere the same, but their routing was disrupted in the absence of Pprotein. Tyrosinase in these aberrant vesicles could be detected by DOPAstaining and was thus enzymatically active. The increase in maturemelanosomes in melan-p1 cells cultured in high tyrosine was notaccompanied by a major reduction in the number of 50 nm vesicles,suggesting partial, but not complete, correction of the p phenotype bytyrosine.

Example 5

[0315] Targeting of Lysosomal Hydrolases in Melan-a and Melan-p Cells

[0316] This experiment demonstrates that melan-p cells do not properlytarget a certain class of lysosomal hydrolases to the lysosome.

[0317] Melan-a and melan-p cells as described above in Example 1 wereseeded to high density and grown in low tyrosine (14 μM) DME medium.Large granule and small granule fractions were prepared and centrifugedon pre-layered sucrose gradients as described in Rosemblat et al., 1994,supra; and Seiji, 1963, Annals N.Y. Acad. Sci., 100:497-533. Fractionswere collected from the top down.

[0318] Appropriate reaction substrates for the lysosomal enzyme assaysprepared in 0.2 M sodium acetate, 1% TritonX-100 were as follows:

[0319] β-hexosaminidase-4 mM4-methylumbelliferyl-N-acetyl-β-D-glucosaminide

[0320] β-glucosidase-4.6 mM 4-methylumbelliferyl-N-acetyl-β-D-glucoside

[0321] β-glucuronidase-4.6 mM4-methylumbelliferyl-N-acetyl-β-D-glucoronide

[0322] β-galactosidase-4.6 mM4-methylumbelliferyl-N-acetyl-β-D-galactoside

[0323] Acid phosphatase-22.5 mM 4-methylumbelliferyl-phosphate

[0324] The reaction mix was prepared in 96 well flat bottom plates. Eachwell was loaded with 25 μl of a gradient fraction, 2.5 μl 1 M sodiumacetate and 27.5 μl of the appropriate substrate mix. The plates werecovered with parafilm and incubated at 37° C. β-hexosaminidase reactionswere incubated for 50 minutes, β-glucosidase, β-glucuronidase andβ-galactosidase reactions were incubated for 20 minutes, and acidphosphatase reactions were incubated for 10 minutes. Reaction wasstopped by addition of 200 μl of stop buffer (132 mM glycine, 68 mMsodium chloride, 83 mM anhydrous sodium carbonate), and plates readimmediately using an excitation wavelength of 370 nm and an emissionwavelength of 460 nm.

[0325] In both melan-a and melan-p cells, very little of the lysosomalhydrolases were detected in the small granule fraction (see FIGS. 8-12).This result was expected because the small granule fraction consisted ofmostly small vesicles in which lysosomal hydrolases do not normallyaccumulate. The large granule fraction contains endoplasmic reticulum,Golgi organelles, lysosomes and melanosomes and, hence, should containmost of the lysosomal hydrolases. With respect to acid phosphatase,there was only slightly less overall activity for the enzyme in largegranule fractions from melan-p cells as compared to those from melan-acells (FIG. 8B). Additionally, there was a minor shift in localizationof acid phosphatase to slightly less dense fractions in the melan-pcells as compared to melan-a cells. However, with respect to the otherlysosomal hydrolases assayed, the differences between melan-a andmelan-p cells was dramatic. In fact, the overall activity ofβ-hexosaminidase, β-glucosidase, β-glucuronidase and β-galactosidase wassignificantly reduced in melan-p cells as opposed to melan-a cells (seeFIGS. 9-12, right panels). This loss of activity could not be attributedto a shift of the enzymes within the cells because whole cell extractsdemonstrated the similar significant decreases in activity ofβ-hexosaminidase, β-glucosidase, β-glucuronidase and β-galactosidase inmelan-p cells as opposed to melan-a cells, but with essentially nodifference in the total amounts of alkaline phosphatase between melan-pand melan-a cells (results not shown). While the same large granulefractions from melan-a cells that contained acid phosphatase alsocontained most of the β-hexosaminidase, β-glucosidase, β-glucuronidaseand β-galactosidase activities, melan-p cells had a significantreduction in activity of these enzymes in the large granule fractions.Thus, β-hexosaminidase, β-glucosidase, β-glucuronidase andβ-galactosidase enzymes do not accumulate correctly in lysosomes inmelan-p cells.

[0326] Unlike acid phosphatase, the enzymes β-hexosaminidase,β-glucosidase, β-glucuronidase and β-galactosidase are not transportedto the cell surface prior to eventually reaching the lysosome. Instead,these enzymes are transported from the trans-Golgi network to the lateendosome via the activity of the M6P/IGF-II receptor. The differences intargeting of these two classes of lysosomal hydrolases in melan-p cellsversus melan-a cells indicates that disruption of P protein functionaffects M6P/IGF-II receptor-mediated targeting. Based on our resultsshowing the secretion of tyrosinase from melan-p cells, and theintracellular depletion of β-hexosaminidase, β-glucosidase,β-glucuronidase and β-galactosidase in large granule fractions from thesame cells, this class of lysosomal enzymes should be secreted from themelan-p cells. Accordingly, targeting of these enzymes, assayed by anincrease in secretion or a reduction in accumulation in lysosomalmembrane fractions, can also be used as part of an assay to screen forcompounds that affect the function of P protein.

Example 6

[0327] Effects of Compounds that Alter Late Endosomal/LysosomalTrafficking

[0328] Experiments were conducted in order to determine the affect onmelanin production of various compounds that alter lateendosomal/lysosomal trafficking. Cells utilized in these studies weremelan-a melanocytes as described above.

[0329] Melan-a melanocytes were incubated in DMEM (10% fetal calf serum,2 mM L-glutamine, 1 mM sodium pyruvate, 1% MEM non-essential amino acids100X, 50 μ/L penicillin, 50 μg/L streptomycin). Immediately before usingthe medium, tetradecanoyl phorbol acetate (TPA) was added at 200 nM.Cells were seeded in T-25 flasks with 4×10⁴ cells/ml×4.5 ml/flask(10-20% confluent) and were grown at 37° C. with 5% CO₂. Twenty-fourhours later, a compound to be tested (diluted in 0.5 ml media) was addedto the media. Forty-eight hours after the addition of drug, both themedia and drug were changed. Cells were harvested after an additional 48or 72 hours (100% confluent).

[0330] In harvesting the cells, the reagents and cells were kept at 4°C. Briefly, the media was removed from the cells, and one milliliter ofmedia was reserved if needed for the tyrosinase assay. The cells werethen rinsed with approximately 500 μl cold 1X phosphate buffered saline(PBS) until the PBS rinse was clear. Cold extraction buffer (50 mM Tris,(pH 7.5), 2 mM EDTA (pH 7.8), 150 mM NACE, and 1% Triton x-100 (Sigma,St. Louis, Mo.), (500 μl) was added, and the sample was allowed toincubate on ice for a few minutes or until cells began to peel off thebottom of the flask. After tapping the flask to encourage the cells tofall off, the 500 μl of the extraction buffer/cells were removed andplaced in a microfuge tube. After spinning the sample for 5 minutes at14,000×g at 4° C., the supernatant was removed and saved in a microfugetube for the protein assay (and tyrosinase assay, if necessary). Thecell pellets were either stored at 4° C. overnight or at −20° C. forlonger periods before assaying for melanin.

[0331] In order to assay for melanin, 300-500 μl of ethanol/ether (1:1)was added to each pellet of cells. The sample was vortexed and allowedto stand for approximately 10 minutes or until precipitated protein wasvisible in the solvent. If necessary, pellets were gently crushed with amicrofuge tube pestle. Care was taken not to break the pellet(s) intomany small pieces which would have made removing the solvent (andleaving melanin behind) difficult. Using a glass pipette, thesolvent/protein was removed, being careful not to remove melanin. Theextraction steps were repeated, and the pellets allowed to dry. Next,250 μl of 2 N NaOH in 20% dimethylsulfoxide (DMSO) was added to eachmicrofuge tube. The samples were heated at 60-70° C. until the melaninwas completely dissolved. For each sample tested, 200 μl of theNaOH/melanin solution was transferred to a 96-well plate. 2 N NaOH in20% DMSO was used as a blank, and the samples were read at a wavelengthof 490 nm. The data were reported as absorbance of melanin per proteincalculated for the total sample.

[0332] In order to determine the effects of progesterone on melaninproduction, melan-a melanocytes were incubated in the presence of 20 μMprogesterone (Sigma, St. Louis, Mo.) as outlined above. In addition,cells were also separately treated with either 300 μM PTU (Sigma, St.Louis, Mo.), a direct inhibitor of tyrosinase, or 100 μM IBMX (Sigma,St. Louis, Mo.), a phosphodiesterase inhibitor.

[0333] The results, as presented in FIG. 13, indicate that progesteronedecreases pigmentation in melan-a melanocytes an average of 52% at 20μM. Other experiments indicated that progesterone decreases pigmentationan average of 45% at 10 μM.

[0334] In order to determine the effects of hydrophobic amines onmelanin production, melan-a melanocytes were incubated in the presenceof 20 μM imipramine (IMP), a cationic amphiphilic drug (Sigma, St.Louis, Mo.) following the procedure outlined above. In addition, cellswere also separately treated with either 300 μM PTU (Sigma, St. Louis,Mo.), or 100 μM IBMX (Sigma, St. Louis, Mo.).

[0335] The results, as presented in FIG. 14, demonstrate that IMPdecreases pigmentation 33% at 20 μM.

[0336] Experiments were also done to test the affects of variousconcentrations of U18666A on melanin production. Results of thisexperiment are presented in FIG. 15 and indicate that U18666A decreasespigmentation an average of 60% as compared to control at a concentrationof 2.5 μM and shows a significant decrease in pigmentation to aconcentration as low as 2.5 nM.

[0337] Experiments were also conducted in order to test several analogcompounds of U18666A for an affect on pigment production. The results ofthis experiment are presented in FIG. 16. Most notable are the U18666Aderivatives CP-352369, CP-598755-01, CP-602367, UK-204039, UK204041, andUK204042 (Pfizer, Inc., Groton, Conn.), which significantly decreasemelanin production in melan-a melanocytes.

[0338] In order to determine if U18666A can reduce the effect of IBMX(Sigma, St. Louis, Mo.) alone, which normally would affect melan-a cellsby increasing pigment production, cells were incubated with thecombination of U18666A and IBMX (Sigma, St. Louis, Mo.). The results arepresented in FIG. 17. U18666A in combination with the phosphodiesteraseinhibitor IBMX (Sigma, St. Louis, Mo.) reduces the effect of IBMX(Sigma, St. Louis, Mo.) alone by 75%.

Example 7

[0339] Exposure of Cells to U 18666A Does Not Alter Tyrosinase Activity

[0340] In order to determine whether the decrease in the amount ofmelanin in cells exposed to the hydrophobic amine U18666A could beattributed to an affect on the activity of tyrosinase, the activity oftyrosinase in cells exposed to U18666A was determined. In theseexperiments, tritiated tyrosine is converted to dopaquinone bytyrosinase, where water is released as a product. By labeling tyrosinewith ³H, tritiated water is released and is a measure of tyrosinaseactivity.

[0341] All samples were assayed in duplicate or triplicate (1 μl³H-tyrosine (³H-Tyr) 46.0 Ci/mmole per sample (Amersham PharmaciaBiotech, Piscataway, N.J.). ³H-Tyr was divided equally among microfugetubes for faster drying. The filled tubes were covered with parafilm andholes were poked through the parafilm with a syringe. The ³H-Tyr wasdried in a speed vac for approximately 15 minutes. The ³H-Tyr was washedtwo times with an equal volume of ethanol. The ³H-Tyr was reconstitutedin 10x its original volume with DOPA (Sigma, St. Louis, Mo.) (0.5 mg/mlDOPA in 0.1 M NaPO₄, pH 6.8). The ³H-Tyr samples were pooled together.Reaction mixtures were prepared in short borosilicate tubes as follows.Briefly, 10 μl ³H-Tyr was added to a 50 μl sample of 0.1 M NaPO₄containing 1% Triton X-100. The sample was incubated for 1 hour at 37°C. in a water bath. To stop the reaction, 1.0 ml of 10% activatedcharcoal in 0.1 M citric acid was added and the sample was vortexedthoroughly. Activated charcoal stops the reaction by absorbingfree-floating organic compounds, for example the ³H-Tyr. Next, thesample(s) were centrifuged for 5 minutes at 700 rpm. Supernatants werecollected (670 μl) and loaded on cationic exchange columns packed withAG 50W-X8 Resin (200-400 mesh) (Bio-Rad Laboratories, Hercules, Calif.).Buffer (0.5 ml of 0.1 M citric acid) was loaded onto the column to washsample through. Ten ml of scintillation fluid was added, and the samplewas vortexed thoroughly. ³H activity was counted in scintillationcounter.

[0342] The results of the experiment, presented in FIG. 18A, indicatethat no significant difference in the total amount of tyrosinaseactivity is found in cells exposed to U18666A compared to the control,untreated cells. These results indicate that the decrease in melaninobserved when melan-a melanocytes are exposed to U18666A cannot beattributed to a decrease in the total amount of tyrosinase in the cells,suggesting that the decrease in melanin is the result of alteredlocalization of tyrosinase.

[0343] In order to confirm the results of the tyrosinase assay, theamounts of tyrosinase protein in control, untreated melan-a melanocytesand U186666A-treated melan-a melanocytes were compared by Western blotanalysis. Standard procedures were followed for protein electrophoresisand protein transfer to a support membrane (see, e.g., MolecularCloning: A Laboratory Manual (1989) Cold Spring Harbor, N.Y., ColdSpring Harbor Laboratory, 2^(nd) edition, Sambrook, J., Fritsch, E. F.,and Maniatis, T.). Briefly, protein samples were run on a 7.5% SDSpolyacrylamide gel overnight at 35 volts. The proteins were transferredfrom the gel onto nitrocellulose over 1.5 hours at 400 mA. Non-specificbinding sites on the membrane were blocked by incubating the membrane in3% milk in phophate buffered saline (PBS) for 1 hour. The membrane wasthen incubated with primary antibody (Pep 7, 1:2000, obtained fromVincent Hearing, NIH, Bethesday, Md.) in 3% milk in PBS overnight at 4°C. The membrane was washed with PBS with 0.05% Tween 20 added (PBS-T)for six times, five minutes each wash. After washing, the membrane wasincubated with secondary antibody (horse radish peroxidase anti-rabbit,1:2000, Amersham Life Science, Piscataway, N.J.) in 3% milk in PBS for 1hour. Following washing for 8 times, 5 minutes each wash, with PBS-T,the membrane was incubated with ECL detection reagent (NEN Life ScienceProducts, Boston, Mass.) for 1 minute. Autoradiography was performedfollowing standard procedures.

[0344] Results of the Western blot analysis are presented in FIG. 18B.This figure shows that the total amount of tyrosinase in the cell afterexposure to U18666A is not significantly less than the control,untreated cell.

Example 8

[0345] Exposure of Cells to U18666A Alters the IntracellularLocalization of Tyrosinase

[0346] In order to determine whether treatment with U18666A affects thecellular localization of tyrosinase, melan-a melanocytes werefractionated by sucrose gradient centrifugation after being exposed toU18666A.

[0347] Large granule and small granule fractions were prepared andcentrifuged on pre-layered sucrose gradients as previously described inSeiji (1963) Annals N.Y. Acad. Sci. 100:497-533.

[0348] Sucrose gradient fractions were collected from the top down, andthe samples were analyzed for tyrosinase activity as previouslydescribed above.

[0349] The results are presented in FIG. 19 (small granulefractionation) and FIG. 8 (large granule fractionation). These dataindicate that the localization of tyrosinase is markedly affected byexposure to U18666A. For example, in FIG. 19 a shift in the distributionof tyrosinase is evident in comparing fraction 5 of the control andU18666A samples. The change in the distribution of tyrosinase is evenmore evident in the large granule fractionation study (see FIG. 20,fractions 6-8, for the large granule fraction control (LGF-C) and largegranule fraction U18666A (LGF-U18)).

[0350] These results support the position that U18666A inhibits melaninproduction by altering the normal cellular pattern of tyrosinaselocalization.

Example 9

[0351] Exposure of Cells to Bafilomycin or Concanamycin

[0352] Wild type melan-a (a/a, P/P), an immortalized melanocyte linederived from C57BL/6J mice; melan-p1 melanocytes (a/a, p^(cp)/p^(25H)),an immortalized line derived from p-null mice; melan-p1 (P10), amelan-p1 line stably transfected with an expression plasmid carrying thepink-eyed dilution gene; B16, an amelanotic mouse melanoma cell line;and B16F10, a slightly pigmented mouse melanoma cell line, were used inthis study.

[0353] Cells were cultured in RMPI-1640 (Sigma, St. Louis, Mo.)supplemented with 10% fetal calf serum, 5% sodium pyruvate, 5%glutamate, 5 units/ml penicillin, 5 μg/ml streptomycin, 1% non-essentialamino acids and 200 nM 12-O-tetradecanoyl phorbol 13-acetate for 24hours. The medium was than replaced with RPMI containing either 0, 1, 2or 2.5 μM bafilomycin A1 (Sigma, St. Louis, Mo.) or 0, 0.5 or 1.0 μMconcanamycin A (Sigma, St. Louis, Mo.). After 36 hours, the medium wasremoved and the cells extracted in 100 μl extraction buffer (1% Triton-X100, 50 mM Tris, 2 mM EDTA, 150 mM NaCl). The samples were centrifugedat 13,000 rmp in a microfuge tube for 30 minutes. The supernatant wasremoved and protein content determined using a protein assay kit(BioRad, Richmond, Calif.).

[0354] The melanin content in the pellet was determined followingremoval of the supernatant. Each sample was treated with 0.5 mlethanol:ether (1:1), vortexed and incubated at room temperature for 10minutes. The solvent was removed and the melanin was washed again withethanol:ether (1:1). Following removal of the second wash, 200 μl 2 Nsodium hydroxide, 20% DMSO was added and the sample heated to 60° C.until the melanin was dissolved. The sample was then transferred to a96-well plate and the optical density was read at 490 nm. The opticaldensity was then normalized to protein concentration in thecorresponding lysate.

[0355]FIGS. 21 and 22 show the results obtained after bafilomycin A1 orconcanamycin A treatment, respectively. FIG. 21 demonstrates that themelanin content of wild type melanocytes (Melan-a) and melanocytes withdiminished or absent P protein activity stably transfected with anexpression plasmid carrying the pink-eyed dilution gene (Melan-p (P10))initially increases in the presence of μM concentrations of bafilomycinA1 but drops at the highest concentration tested. Melanin levels in ap-null line (Melan-p) increased with an increase in bafilomycin A1concentration. A melanoma line lacking tyrosinase and P protein (B16)does not respond to bafilomycin A, while a second melanoma line whichhas detectable levels of tyrosinase, but not P protein (b16F-10)demonstrates increasing pigmentation which diminishes at higherconcentrations.

[0356]FIG. 22 shows that the melanin content of wild type melanocytes(Melan-a) and melanocytes with diminished or absent P protein activitystably transfected with an expression plasmid carrying the pink-eyeddilution gene (Melan-p (P10)) is decreased in the presence of μMconcentrations of concanamycin A, while melanin levels in a p-null line(Melan-p) increase with an increase in concanamycin A concentration. Amelanoma line lacking tyrosinase and P protein (B16) does not respond toconcanamycin A, while a second melanoma line which has detectable levelsof tyrosinase, but not P protein (B16F10) demonstrates increasingpigmentation which diminishes at higher concentrations.

[0357] These results indicate that melanogenesis is activated in p-nullmelanogenesis by exposure to the ATPase inhibitors bafilomycin and/orconcanamycin.

[0358] Equivalents

[0359] The foregoing written specification is sufficient to enable oneskilled in the art to practice the invention. Indeed, variousmodifications of the above-described means for carrying out theinvention which are obvious to those skilled in the field of molecularbiology, medicine or related fields are intended to be within the scopeof the following claims.

What is claimed is:
 1. A method of screening for compounds that inhibitmelanogenesis, the method comprising: treating cells expressing atyrosinase-encoding gene with a test compound, and determining thecellular localization of tyrosinase in the presence of the testcompound; wherein a change in the cellular localization of tyrosinase inthe presence of the test compound as compared to in the absence of thetest compound indicates that the test compound is a candidate for acompound that inhibits melanogenesis.
 2. The method of claim 1, whereinthe cells further express a P protein-encoding gene, and wherein thechange in the cellular localization of tyrosinase in the presence of thetest compound as compared to in the absence of the test compound isdependent upon the expression of the P protein-encoding gene.
 3. Themethod of claim 1 or 2, wherein the cellular localization of tyrosinaseis determined by assaying the amount of tyrosinase secreted by the cellsin the presence of the compound, wherein an increase in the amount oftyrosinase secreted by the cells in the presence of the test compound ascompared to in the absence of the test compound indicates that the testcompound is a candidate for a compound that inhibits melanogenesis. 4.The method of claim 1 or 2, wherein the cellular localization oftyrosinase is detected by assaying for tyrosinase activity.
 5. Themethod of claim 1 or 2, wherein the cellular localization of tyrosinaseis detected by assaying for the presence of tyrosinase protein usingimmunological techniques.
 6. The method of claim 1 or 2 furthercomprising the step of assaying the amount of tyrosinase associated in ahigh molecular weight complex in the presence of the test compound,wherein a decrease in the amount of tyrosinase associated in a highmolecular weight complex in the presence of the test compound ascompared to in the absence of the test compound indicates that the testcompound is a candidate for a compound that inhibits melanogenesis. 7.The method of claim 1 or 2 further comprising the step of assaying theamount of TRP-1 or TRP-2 protein associated in a high molecular weightcomplex in the presence of the compound, wherein a decrease in theamount of TRP-1 or TRP-2 protein associated in a high molecular weightcomplex in the presence of the test compound as compared to in theabsence of the test compound indicates that the test compound is acandidate for a compound that inhibits melanogenesis.
 8. The method ofclaim 1 or 2 further comprising the step of assaying the number or sizeof melanosomes in the cells in the presence of the compound, wherein adecrease in the number or size of melanosomes in the cells in thepresence of the test compound as compared to in the absence of the testcompound indicates that the test compound inhibits melanogenesis.
 9. Themethod of claim 1 or 2 further comprising the step of assaying the massor length of tyrosinase in the cells in the presence of the compound,wherein a decrease in the mass or length of tyrosinase in the cells inthe presence of the test compound as compared to in the absence of thetest compound indicates that the test compound is a candidate for acompound that inhibits melanogenesis.
 10. The method of claim 1 or 2further comprising the step of assaying for the levels and/or targetingof lysosomal hydrolases in the cells in the presence of the compound,wherein a decrease in accumulation of lysosomal hydrolases that aretransported via the M6P/IGF-II receptor in the lysosome in the cells inthe presence of the test compound as compared to in the absence of thetest compound indicates that the test compound is a candidate for acompound that inhibits melanogenesis.
 11. The method of claim 1 or 2,wherein the cells are grown in the presence of low tyrosine.
 12. Themethod of claim 11 wherein the concentration of tyrosine is 0.01-0.03mM.
 13. The method of claim 1 wherein the cells are melanocytes.
 14. Themethod of claim 1 wherein the cells are melanoma cells.
 15. The methodof claim 1 wherein the cells are derived from a mammal.
 16. The methodof claim 15 wherein the mammal is a human.
 17. The method of claim 15wherein the mammal is selected from the group consisting of mouse,hamster, and guinea pig.
 18. A method of screening for compounds thatincrease melanogenesis comprising: treating cells expressing atyrosinase-encoding gene with a test compound, and determining theamount of tyrosinase secreted by the cells in the presence of the testcompound; wherein a decrease in the amount of tyrosinase secreted by thecells in the presence of the test compound as compared to in the absenceof the test compound indicates that the test compound is a candidate fora compound that increases melanogenesis.
 19. The method of claim 18wherein the cells further express a P protein-encoding gene, and whereinthe decrease in the amount of tyrosinase secreted by the cells in thepresence of the test compound as compared to in the absence of the testcompound is dependent upon the expression of the P protein-encodinggene.
 20. The method of claim 18 or 19 further comprising determining aratio of tyrosinase inside the cells to tyrosinase secreted by thecells, wherein an increase in the ratio in the presence of the testcompound as compared to in the absence of the test compound indicatesthat the test compound induces melanogenesis.
 21. The method of claim 18or 19, wherein the amount of tyrosinase is detected by assaying fortyrosinase activity.
 22. The method of claim 18 or 19, wherein theamount of tyrosinase is detected by assaying for the presence oftyrosinase protein using immunological techniques.
 23. The method ofclaim 18 wherein the cells are melanocytes.
 24. The method of claim 18wherein the cells are melanoma cells.
 25. The method of claim 23 or 24,wherein the cells are visually examined for an increase in melaninproduction.
 26. The method of claim 23 or 24 wherein the cells do notexpress P protein, and wherein a decrease in the amount of tyrosinasesecreted by the cells in the presence of the test compound as comparedto in the absence of the test compound indicates that the test compoundis a candidate for a compound that mimics P protein function.
 27. Themethod of claim 23 wherein the cells are mouse melan-p melanocytes. 28.The method of claim 18 wherein the cells are derived from a mammal. 29.The method of claim 28 wherein the mammal is a human.
 30. The method ofclaim 28 wherein the mammal is selected from the group consisting ofmouse, hamster, and guinea pig.
 31. The method of claim 26, wherein thecells are visually examined for an increase in melanin production.
 32. Amethod of screening for compounds that affect the function of P protein,the method comprising: contacting a system with a test compound, thesystem comprising P protein and tyrosinase; and identifying those testcompounds that affect tyrosinase activity in the system in a Pprotein-dependent manner.
 33. The method of claim 32 wherein the systemis a cell that expresses a P protein-encoding gene and atyrosinase-encoding gene.
 34. The method of claim 33 wherein the cell isa cultured cell.
 35. The method of claim 32 wherein compounds thatdecrease tyrosinase activity in the system are identified as compoundsthat inhibit the function of P protein.
 36. The method of claim 35further comprising the step of assaying for the targeting of lysosomalhydrolases in the cells in the presence of the compound, wherein adecrease in accumulation of lysosomal hydrolases that are transportedvia the M6P/IGF-II receptor in the lysosome in the cells in the presenceof the test compound as compared to in the absence of the test compoundindicates that the test compound inhibits the function of P protein. 37.The method of claim 32 wherein compounds that result in an increase intyrosinase activity in the system are identified as compounds thatincrease the function of P protein.
 38. The method of claim 32 whereinthe P protein-encoding gene is derived from a mammal selected from thegroup consisting of human, hamster, guinea pig, and mouse.
 39. Themethod of claim 32 wherein the tyrosinase-encoding gene is derived froma mammal selected from the group consisting of human, hamster, guineapig, and mouse.
 40. A method of screening for compounds that affect thefunction of P protein, the method comprising: using a primary screeningmethod to preliminarily determine whether a test compound may affect Pprotein function; and using one or more secondary screening methods todetermine whether the test compound affects P protein function.
 41. Themethod of claim 40, wherein the primary screening method comprises atleast one screening assay selected from the group consisting of assayingfor secretion of tyrosinase and assaying for the missorting of at leastone lysosomal hydrolase.
 42. A method of screening for compounds thataffect the function of P protein, the method comprising: modeling acompound that affects the function of the P protein; making chemicalanalogs of the compound; and assaying the chemical analogs for theireffect on the function of P protein.
 43. The method of claim 42 whereinthe compound is imipramine.
 44. A method of decreasing melaninproduction in a melanocyte, comprising contacting the melanocyte with aneffective amount of a compound that effects an alteration in lateendosomal/lysosomal trafficking in the melanocyte, wherein thealteration in late endosomal/lysosomal trafficking results in a decreasein melanin production in the melanocyte.
 45. The method of claim 44,wherein the alteration in late endosomal/lysosomal trafficking iseffected by contacting the melanocyte with a compound that is anantagonist of late endosomal/lysosomal trafficking.
 46. The method ofclaim 44, wherein the alteration in late endosomal/lysosomal traffickingis an alteration in late endosomal/lysosomal cholesterol trafficking.47. The method of claim 44, wherein the alteration in lateendosomal/lysosomal trafficking is effected by contacting the melanocytewith a compound selected from the group consisting of (a) progesterone,(b) a hydrophobic amine, (c) sphingosine, and (d) a compound of theformula

wherein X is O or S; R₁ is —C(O)(C₁-C₆)alkyl or—(CH₂)_(n)—O—(C₁-C₆)alkyl, or —(CH₂)_(n)—NR₇R₈ where n is 0-3, and whereeach of R₇ and R₈ are independently selected from H and (C₁-C₆)alkyl; R₂is H or (C₁-C₆)alkyl; R₃ is H or (C₁-C₆)alkyl; R₄ is —C(O)(C₁-C₆)alkyl;R₅ is H or —(C₁-C₆)alkyl; or R₄ and R₅ together are ═O; and R₆ is H or—(C₁-C₆)alkyl or —(CH₂)_(n)—NR₉R₁₀ where each of R₉ and R₁₀ areindependently selected from H and (C₁-C₆)alkyl.
 48. The method of claim47, wherein the compound is progesterone.
 49. The method of claim 47,wherein the compound is a hydrophobic amine.
 50. The method of claim 49,wherein the hydrophobic amine is selected from the group consisting of aphenothiazine, and a tricyclic antidepressant.
 51. The method of claim50, wherein the compound is a phenothiazine.
 52. The method of claim 51,wherein the phenothiazine is selected from the group consisting oftrifluoperazine, chlorpromazine, prochlorperazine, triflupromazine,promazine, thioridazine, mesoridaine, piperacetazine, perphenazine,fluphenazine, acetophenazine, and thiethylperazine.
 53. The method ofclaim 50, wherein the compound is a tricyclic antidepressant.
 54. Themethod of claim 53, wherein the tricyclic antidepressant is selectedfrom the group consisting of imipramine, nortriptyline, protriptyline,trimipramine, and doxepin.
 55. The method of claim 47, wherein thecompound is sphingosine.
 56. The method of claim 47, wherein thecompound is selected from the group consisting of:

or a pharmaceutically acceptable salt or solvate thereof.
 57. A methodof reducing skin pigmentation, comprising contacting skin with apharmaceutically effective amount of a compound that effects analteration in late endosomal/lysosomal trafficking, wherein analteration in late endosomal/lysosomal trafficking results in areduction of skin pigmentation.
 58. The method of claim 57, wherein analteration in late endosomal/lysosomal trafficking is effected bycontacting the skin with a compound that is an antagonist of lateendosomal/lysosomal trafficking.
 59. The method of claim 57, wherein thealteration in late endosomal/lysosomal trafficking is an alteration inlate endosomal/lysosomal cholesterol trafficking.
 60. The method ofclaim 57, wherein the alteration in late endosomal/lysosomal traffickingis effected by contacting the skin with a pharmaceutically effectiveamount of a compound selected from the group consisting of (a)progesterone, (b) a hydrophobic amine, (c) sphingosine, and (d) acompound of the formula

wherein X is O or S; R₁ is —C(O)(C₁-C₆)alkyl or—(CH₂)_(n)—O—(C₁-C₆)alkyl, or —(CH₂)_(n)—NR₇R₈ where n 0-3, and whereeach of R₇ and R₈ are independently selected from H and (C₁-C₆)alkyl; R₂is H or (C₁-C₆)alkyl; R₃ is H or (C₁-C₆)alkyl; R₄ is —C(O)(C₁-C₆)alkyl;R₅ is H or —(C₁-C₆)alkyl; or R₄ and R₅ together are ═O; and R₆ is H or—(C₁-C₆)alkyl or —(CH₂)_(n)—NR₉R₁₀ where each of R₉ and R₁₀ areindependently selected from H and (C₁-C₆)alkyl.
 61. The method of claim60, wherein the compound is progesterone.
 62. The method of claim 60,wherein the compound is a hydrophobic amine.
 63. The method of claim 62,wherein the hydrophobic amine is selected from the group consisting of aphenothiazine, and a tricyclic antidepressant.
 64. The method of claim63, wherein the compound is a phenothiazine.
 65. The method of claim664, wherein the phenothiazine is selected from the group consisting oftrifluoperazine, chlorpromazine, prochlorperazine, triflupromazine,promazine, thioridazine, mesoridaine, piperacetazine, perphenazine,fluphenazine, acetophenazine, and thiethylperazine.
 66. The method ofclaim 63, wherein the compound is a tricyclic antidepressant.
 67. Themethod of claim 66, wherein the tricyclic antidepressant is selectedfrom the group consisting of imipramine, nortriptyline, protriptyline,trimipramine, and doxepin.
 68. The method of claim 60, wherein thecompound is sphingosine.
 69. The method of claim 60, wherein thecompound is selected from the group consisting of:


70. A pharmaceutical composition for reducing skin pigmentation,comprising a skin pigmentation reducing effective amount of a compoundthat effects an alteration in late endosomal/lysosomal trafficking in askin cell and a pharmaceutically acceptable carrier.
 71. The compositionof claim 70, wherein the alteration in late endosomal/lysosomaltrafficking is effected by contacting the melanocyte with a compoundthat is an antagonist of late endosomal/lysosomal trafficking.
 72. Thecomposition of claim 70, wherein the alteration in lateendosomal/lysosomal trafficking is an alteration in lateendosomal/lysosomal cholesterol trafficking.
 73. The composition ofclaim 70, wherein the compound that effects the alteration in lateendosomal/lysosomal trafficking is selected from the group consisting of(a) progesterone, (b) a hydrophobic amine, (c) sphingosine, and (d) acompound of the formula

wherein X is O or S; R₁ is —C(O)(C₁-C₆)alkyl or—(CH₂)_(n)—O—(C₁-C₆)alkyl, or —(CH₂)_(n)—NR₇R₈ where n is 0-3, and whereeach of R₇ and R₈ are independently selected from H and (C₁-C₆)alkyl; R₂is H or (C₁-C₆)alkyl; R₃ is H or (C₁-C₆)alkyl; R₄ is —C(O)(C₁-C₆)alkyl;R₅ is H or —(C₁-C₆)alkyl; or R₄ and R₅ together are ═O; and R₆ is H or—(C₁-C₆)alkyl or —(CH₂)_(n)—NR₉R₁₀ where each of Rg and R₁₀ areindependently selected from H and (C₁-C₆)alkyl.
 74. The composition ofclaim 73, wherein the compound is progesterone.
 75. The composition ofclaim 73, wherein the compound is a hydrophobic amine.
 76. Thecomposition of claim 73, wherein the hydrophobic amine is selected fromthe group consisting of a phenothiazine, and a tricyclic antidepressant.77. The composition of claim 76, wherein the compound is aphenothiazine.
 78. The composition of claim 77, wherein thephenothiazine is selected from the group consisting of trifluoperazine,chlorpromazine, prochlorperazine, triflupromazine, promazine,thioridazine, mesoridaine, piperacetazine, perphenazine, fluphenazine,acetophenazine, and thiethylperazine.
 79. The composition of claim 76,wherein the compound is a tricyclic antidepressant.
 80. The compositionof claim 79, wherein the tricyclic antidepressant is selected from thegroup consisting of imipramine, nortriptyline, protriptyline,trimipramine, and doxepin.
 81. The composition of claim 73, wherein thecompound is sphingosine.
 82. The composition of claim 73, wherein thecompound is selected from the group consisting of:


83. A method of activating melanogenesis comprising contacting amelanocyte with diminished or absent P protein activity with apharmaceutically effective amount of a compound that inhibits ATPase,whereby the inhibition of ATPase results in an activation ofmelanogenesis in the melanocyte with diminished or absent P proteinactivity.
 84. The method according to claim 83, wherein the melanocytewith diminished or absent P protein activity is contacted with apharmaceutically effective amount of a compound selected from the groupconsisting of bafilomycin, concanamycin, and derivatives thereof. 85.The method according to claim 83, wherein the melanocyte with diminishedor absent P protein activity is contacted with a pharmaceuticallyeffective amount of bafilomycin or a derivative thereof.
 86. The methodaccording to claim 83, wherein the melanocyte with diminished or absentP protein activity is contacted with a pharmaceutically effective amountof concanamycin or a derivative thereof.
 87. A method of treatingtyrosinase-positive, oculocutaneous albinism in an individual in needthereof, the method comprising contacting skin of the individual with apharmaceutically effective amount of a compound that inhibits ATPase,whereby the inhibition of ATPase results in an activation ofmelanogenesis in the individual.
 88. The method according to claim 87,wherein the skin of the subject is contacted with a pharmaceuticallyeffective amount of a compound selected from the group consisting ofbafilomycin or a derivative thereof and concanamycin or a derivativethereof.
 89. The method according to claim 88, wherein the skin of thesubject is contacted with a pharmaceutically effective amount ofbafilomycin or a derivative thereof.
 90. The method according to claim88, wherein the skin of the subject is contacted with a pharmaceuticallyeffective amount of concanamycin or a derivative thereof.
 91. A kitcomprising a pharmaceutically effective amount of a compound thatmodulates melanogenesis by affecting P protein function, inhibiting lateendosomal/lysosomal trafficking, or inhibiting an ATPase.
 92. The kit ofclaim 91, further comprising a set of written instructions describinghow to use the compound to modulate skin pigmentation.